EPA/6Q0/R-21/059 | September 2021
www.epa.gov/emergency-response-research
United States
Environmental Protection
Agency
&EPA
Development of Rapid Viability-Reverse
Transcriptase PCR (RV-RT-PCR) Method
for Detection of Infectious SARS-CoV-2
from Environmental Samples
Office of Research and Development
Homeland Security Research Program

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EPA/600/ R-21/059
September 2021
Development of Rapid Viability-Reverse Transcriptase PCR
(RV-RT-PCR) Method for Detection of Infectious SARS-CoV-2
from Environmental Samples
By
Sanjiv R. Shah, Ph.D.
U.S. Environmental Protection Agency
Washington, DC
And
Staci Kane, Ph.D.
Maher Elsheikh, Ph.D.
Teneile Alfaro
Lawrence Livermore National Laboratory
United States Department of Energy
Livermore, CA 94551
EPA IA DW-089-92527601 - 0

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Disclaimer
U.S. Environmental Protection Agency
The United States Environmental Protection Agency (U.S. EPA) through its Office of Research and
Development funded and managed the research described here (EPA IA DW-089-92527601 - 0). This
report has been reviewed and approved for public release in accordance with the policies of the U.S.
EPA. Note that approval does not signify that the contents necessarily reflect the views of the Agency.
Mention of trade names, products, or services does not convey EPA approval, endorsement, or
recommendation.
Lawrence Livermore National Laboratory
This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore
National Laboratory under Contract DE-AC52-07NA27344.
This document was prepared as an account of work sponsored by the Environmental Protection Agency
of the United States government. Neither the United States government nor Lawrence Livermore
National Security, LLC, nor any of their employees makes any warranty, expressed or implied, or
assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any
information, apparatus, product, or process disclosed, or represents that its use would not infringe
privately owned rights. Reference herein to any specific commercial product, process, or service by
trade name, trademark, manufacturer, or otherwise does not constitute or imply its endorsement,
recommendation, or favoring by the United States government or Lawrence Livermore National
Security, LLC. The views and opinions of authors expressed herein do not necessarily state or reflect
those of the United States government or Lawrence Livermore National Security, LLC, and shall not be
used for advertising or product endorsement purposes.
Questions concerning this document, or its application should be addressed to:
Sanjiv R. Shah, Ph.D.
Disaster Characterization Branch
Homeland Security and Materials Management Division
Center for Environmental Solutions and Emergency Response
Office of Research and Development
U.S. Environmental Protection Agency
1300 Pennsylvania Avenue, NW
USEPA-8801RR
Washington, DC 20460
(202) 564-9522
shah, sani iv@epa. gov
If you have difficulty accessing these PDF documents, please contact
McCall.Amelia@epa.gov for assistance.

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Foreword
The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the nation's
land, air, and water resources. Under a mandate of national environmental laws, the Agency strives to
formulate and implement actions leading to a compatible balance between human activities and the
ability of natural systems to support and nurture life. To meet this mandate, EPA's research program is
providing data and technical support for solving environmental problems today and building a science
knowledge base necessary to manage our ecological resources wisely, understand how pollutants affect
our health, and prevent or reduce environmental risks in the future.
The EPA's Center for Environmental Solutions and Emergency Response (CESER) within the Office of
Research and Development (ORD) conducts applied, stakeholder-driven research and provides
responsive technical support to help solve the nation's environmental challenges. The Center's research
focuses on innovative approaches to address environmental challenges associated with the built
environment. We develop technologies and decision-support tools to help safeguard public water
systems and groundwater, guide sustainable materials management, remediate sites from traditional
contamination sources and emerging environmental stressors, and address potential threats from
terrorism and natural disasters. CESER collaborates with both public and private sector partners to
foster technologies that improve the effectiveness and reduce the cost of compliance, while anticipating
emerging problems. We provide technical support to EPA regions and programs, states, tribal nations,
and federal partners, and serve as the interagency liaison for EPA in homeland security research and
technology. The Center is a leader in providing scientific solutions to protect human health and the
environment.
At the onset of the COVID-19 pandemic, the EPA-ORD's Homeland Security Research Program
expanded their research horizons to respond to this public health emergency. To support and expedite
the studies on understanding the surface transmission of SARS-CoV-2 and environmental
epidemiological investigations, a Rapid Viability-Reverse Transcriptase Polymerase Chain Reaction
(RV-RT-PCR) method to detect infectious virus was developed. This report describes the method
development research. The RV-RT-PCR method developed in this project will allow detection of
infectious SARS-CoV-2 in environmental surfaces and any other sample types within hours, rather than
several days taken by the currently used traditional cell-culture-based methods.
Gregory Sayles, Director
Center for Environmental Solutions and Emergency Response

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Table of Contents
Disclaimer	ii
U.S. Environmental Protection Agency	ii
Lawrence Livermore National Laboratory	ii
Foreword	iii
Acronyms and Abbreviations	ix
Trademarked Products	xi
Acknowledgments	xii
Executive Summary	xiv
1.0 Introduction	1
2.0 Materials and Methods	3
2.1	Cell Culture and Viral Propagation	3
2.2	Preparation of Viral Stocks	4
2.3	Median Tissue Culture Infectious Dose (TCID50) Analysis	4
2.4	Rapid-Viability - Reverse Transcriptase PCR Method	5
2.5	RV-RT-PCR Experiments with Viral Suspension	6
2.6	RV-RT-PCR Experiments with Swab Swatches	6
2.7	RV-RT-PCR Experiments with Swabs for SARS-CoV-2	7
2.8	RNA Extraction	8
2.9	MHV Reverse Transcriptase PCR Analysis	8
2.10	SARS-CoV-2 Reverse Transcriptase PCR Analysis	9
2.11	RV-RT-PCR Data Analysis and Interpretation	11
2.12	Biosafety	12
3.0 Quality Assurance and Quality Control	13
3.1	Laboratory Inspections	13
3.2	Calibration	13
3.3	Storage Conditions	13
3.4	Spiking	13
3.5	Real-time PCR Analysis	13
3.6	Replication	14
3.7	Controls	14
3.8	Data Quality Objectives/Data Quality Indicators	14
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3.9 Audit of Data Quality	14
4.0 Results and Discussion	15
4.1	Optimization of growth medium/growth conditions, and monolayer growth of the cell
line to be used as host for the surrogate virus, mouse hepatitis virus (MHV)	16
4.1.1	Preparation of MHV stocks and quantitation by TCID50 analysis	16
4.1.2	Optimal Cell Seeding Density Determination	16
4.2	Optimization of incubation conditions and time intervals to determine an initial method
sensitivity of detection for MHV via RV-RT-PCR, as compared to a traditional tissue
culture infective dose (TCID50) assay, for virus from liquid suspension and recovered
from spiked swab samples	17
4.2.1	RNA Extraction and RT-PCR Analysis from MHV Stock	17
4.2.2	RT-PCR Analysis of MHV-lnfected Cells: RNA Extraction in Tubes	19
4.2.3	RT-PCR Analysis of MHV-lnfected Cells: RNA Extraction in the Plate	20
4.2.4	Viral Concentration Using Amicon Ultrafiltration Devices	22
4.2.5	RV-RT-PCR experiments with MHV stock dilutions	22
4.2.6	RV-RT-PCR analysis of MHV-spiked swab swatches	28
4.3	Evaluation and optimization of growth conditions in a multi-well plate format, and
monolayer and suspension growth of the host cell line for the SARS-CoV-2 virus	35
4.3.1	Establishing Optimal Vero E6 Cell Preparation Procedures for SARS-CoV-2
Propagation	35
4.3.2	Evaluation of Suspension Growth of Vero E6 Cell Cultures	35
4.4	Optimization of virus infection period and post-infection incubation period to determine
an initial method sensitivity of detection of SARS-CoV-2 via RV-RT-PCR, for virus from
liquid suspension and recovered from spiked swab samples	36
4.4.1	Evaluation of SARS-CoV-2 RT-PCR Assays	36
4.4.2	RV-RT-PCR experiments with SARS-CoV-2 dilutions	38
4.4.3	RV-RT-PCR analysis of SARS-CoV-2-spiked swab swatches	47
4.4.4	Comparison of RV-RT-PCR and TCID50 analyses from the same SARS-CoV-
2-spiked swab swatch samples (side-by-side analysis)	56
4.4.5	Analysis of SARS-CoV-2-spiked swab samples using the final optimized RV-
RT-PCR method	61
5.0 Summary and Conclusions	75
6.0 References	77
7.0 Appendix A. Protocol for Rapid Viability Reverse Transcriptase Polymerase Chain
Reaction (RV-RT-PCR)-Based Detection of SARS-CoV-2 from Swab Samples	81
V

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Table of Figures
Figure 1 RV-RT-PCR Method Flow Chart 	5
Figure 2 Schematic of RT-PCR Response Curves from RV-RT-PCR Analysis of a Swab
Sample	15
Figure 3 RT-PCR Results for MHV-extracted RNA with Initial Conditions	18
Figure 4 RT-PCR Results for MHV-extracted RNA with Modified RT-PCR Conditions	19
Figure 5 Average RT-PCR Results for RNA from Triplicate MHV-infected Cell Culture Wells 20
Figure 6 RT-PCR Results for RNA (MHV + 17CI-1) Dilutions	21
Figure 7 Average RT-PCR Results Using N1 and N2 Assays with Synthetic SARS-CoV-2
RNA	38
Figure 8 SARS-CoV-2 RV-RT-PCR Analysis Flow Chart and Estimated Timeline for Swab
Samples	76
Table of Tables
Table 1 Nucleotide Sequences of the Real-time RT-PCR Assay Primer/Probe Set for MHV	8
Table 2 Modified One Step RT-PCR Mix for MHV nsp2 Assay	9
Table 3 Real-time PCR Assays Used for SARS-CoV-2	 10
Table 4 Nucleotide Sequences of the Real-time PCR Assay Primer/Probe Sets for SARS-
CoV-2	10
Table 5 One Step RT-PCR Mix for the SARS-CoV-2 N1 and N2 Assays	11
Table 6 Seeding Densities of 17CI-1 Cells for Different Applications	17
Table 7 RT-PCR Results of RNA from MHV-infected 17CI-1 Cells from 96-well Plates with
Individual Wells Contents Processed in 1.5 ml_ Tubes	20
Table 8 RT-PCR Results of RNA from MHV-infected 17CI-1 Cells from 96-well Plates with
Processing in the Plate	21
Table 9 MHV TCID50 Results with and without Concentration by 10-kDa Ultrafiltration (UF) ..22
Table 10 RV-RT-PCR Results for MHV-infected 17CI-1 Cells with 4-hr Infection	23
Table 11 RV-RT-PCR Results for MHV-infected 17CI-1 Cells with 2-hr Infection	24
Table 12 RV-RT-PCR Results for MHV-infected 17CI-1 Cells with 4-hr and 2-hr Infection -
Replicate Experiment	25
Table 13 Combined RV-RT-PCR Results from Replicate Experiments for MHV-infected
17CI-1 Cells with 4-hr Infection	26
Table 14 Combined RV-RT-PCR Results from Replicate Experiments for MHV-infected
17CI-1 Cells with 2-hr Infection	27
Table 15 MHV Recovery from Swab Swatch Samples	29
Table 16 RV-RT-PCR Results for MHV-Spiked Swab Swatches Processed and Used to
Infect 17CI-1 Cells with 4-hr Infection	30
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Table 17 Summary of RV-RT-PCR Results for MHV-Spiked Swab Swatches Processed
and Used to Infect 17CI-1 Cells with 4-hr Infection	31
Table 18 MHV Recovery from Swab Swatch Samples - Replicate Experiment	32
Table 19 RV-RT-PCR Results for MHV-Spiked Swab Swatches Processed and Used to
Infect 17CI-1 Cells with 4-hr Infection - Replicate Experiment	33
Table 20 Summary of RV-RT-PCR Results for MHV-Spiked Swab Swatches Processed
and Used to Infect 17CI-1 Cells with 4-hr Infection - Replicate Experiment	34
Table 21 Combined RV-RT-PCR Results from Replicate Experiments for MHV-Spiked
Swab Swatches Processed and Used to Infect 17CI-1 Cells with 4-hr Infection and 8-hr
Post-Infection Incubation	34
Table 22 CDC Real-time PCR Assays for SARS-CoV-2 Detection	36
Table 23 One Step RT-PCR Mix for SARS-CoV-2 N1 and N2 Assays Modified from CDC	37
Table 24 RT-PCR Results for SARS-CoV-2 N1 Assay with Synthetic RNA	37
Table 25 RT-PCR Results for SARS-CoV-2 N2 Assay with Synthetic RNA	38
Table 26 RV-RT-PCR Results for SARS-CoV-2-lnfected Vero E6 Cells with 2-hr Infection	40
Table 27 RV-RT-PCR Results for SARS-CoV-2-lnfected Vero E6 Cells with 1-hr Infection -
Second Experiment	42
Table 28 RV-RT-PCR Results for SARS-CoV-2-lnfected Vero E6 Cells with 2-hr Infection -
Second Experiment	43
Table 29 RV-RT-PCR Results for SARS-CoV-2-lnfected Vero E6 Cells with 2-hr Infection -
Third Experiment	45
Table 30 RV-RT-PCR Results for SARS-CoV-2-lnfected Vero E6 Cells with 1-hr Infection -
Third Experiment	46
Table 31 Summary of RV-RT-PCR Avg. ACt Results from Replicate Experiments* for
SARS-CoV-2-lnfected Vero E6 Cells with 2-hr Infection	47
Table 32 SARS-CoV-2 Recovery from Swab Swatch Samples	48
Table 33 RV-RT-PCR Results for SARS-CoV-2-Spiked Swab Swatches Processed and
Used to Infect Vero E6 Cells with 2-hr Infection	49
Table 34 Summary of RV-RT-PCR Results for SARS-CoV-2-Spiked Swab Swatches
Processed and Used to Infect Vero E6 Cells with 2-hr Infection	51
Table 35 SARS-CoV-2 Recovery from Swab Swatch Samples	52
Table 36 RV-RT-PCR Results for SARS-CoV-2-Spiked Swab Swatches Processed and
Used to Infect Vero E6 Cells with 2-hr Infection - Replicate Experiment	53
Table 37 Summary of RV-RT-PCR Results for SARS-CoV-2-Spiked Swab Swatches
Processed and Used to Infect Vero E6 Cells with 2-hr Infection	55
Table 38 Recovery Efficiency of SARS-CoV-2 from Swab Swatches	56
Table 39 Side-by-Side TCID50 and RV-RT-PCR Results for SARS-CoV-2-Spiked Swab
Swatches Processed and Used to Infect Vero E6 Cells with 2-hr Infection	57
Table 40 Summary of Side-by-Side TCID50 and RV-RT-PCR Analyses for SARS-CoV-2-
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Spiked Swab Swatches Processed and Used to Infect Vero E6 Cells with 2-hr Infection..58
Table 41 Recovery Efficiency of SARS-CoV-2 from Swab Swatches - Replicate
Experiment	59
Table 42 Side-by-Side TCID50 and RV-RT-PCR Results for SARS-CoV-2-Spiked Swab
Swatches Processed and Used to Infect Vero E6 Cells with 2-hr Infection - Replicate
Experiment	60
Table 43 Recovery Efficiency of SARS-CoV-2 from Swabs	62
Table 44 RV-RT-PCR Results (N1 Assay) for SARS-CoV-2-Spiked Swabs Processed and
Used to Infect Vero E6 Cells with 2-hr Infection	63
Table 45 RV-RT-PCR Results (N2 Assay) for SARS-CoV-2-Spiked Swabs Processed and
Used to Infect Vero E6 Cells with 2-hr Infection	65
Table 46 Summary of RV-RT-PCR Avg. Ct and ACt Results for SARS-CoV-2-Spiked
Swabs Processed and Used to Infect Vero E6 Cells with 2-hr Infection - N1 Assay	67
Table 47 Summary of RV-RT-PCR Avg. Ct and ACt Results for SARS-CoV-2-Spiked
Swabs Processed and Used to Infect Vero E6 Cells with 2-hr Infection - N2 Assay	68
Table 48 Recovery Efficiency of SARS-CoV-2 from Swabs - Replicate Experiment	69
Table 49 RV-RT-PCR Results (N1 Assay) for SARS-CoV-2-Spiked Swabs Processed and
Used to Infect Vero E6 Cells with 2-hr Infection -Replicate Experiment	70
Table 50 RV-RT-PCR Results (N2 Assay) for SARS-CoV-2-Spiked Swabs Processed and
Used to Infect Vero E6 Cells with 2-hr Infection - Replicate Experiment	71
Table 51 Summary of RV-RT-PCR Avg. Ct and ACt Results for SARS-CoV-2-Spiked
Swabs Processed and Used to Infect Vero E6 Cells with 2-hr Infection (N1 Assay) -
Replicate Experiment	72
Table 52 Summary of RV-RT-PCR Avg. Ct and ACt Results for SARS-CoV-2-Spiked
Swabs Processed and Used to Infect Vero E6 Cells with 2-hr Infection (N2 Assay) -
Replicate Experiment	73
Table 53 Combined RV-RT-PCR Results from Replicate Experiments for SARS-CoV-2-
Spiked Swabs Processed and Used to Infect Vero E6 Cells with 2-hr Infection and 9-hr
Post-Infection Incubation - N1 Assay	74
Table 54 Combined RV-RT-PCR Results from Replicate Experiments for SARS-CoV-2-
Spiked Swabs Processed and Used to Infect Vero E6 Cells with 2-hr Infection and 9-hr
Post-Infection Incubation - N2 Assay	74
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Acronyms and Abbreviations
ABI:	Applied Biosystems Inc.
ATCC:	American Type Culture Collection
Avg.:	average
BEI Resources:	Biodefense and Emerging Infections Research Resources Repository
BHQ: 	Black Hole Quencher
bp: 	Base pair
BSC: 	Biosafety Cabinet
BSL-2: 	BioSafety Level 2
BSL-3: 	BioSafety Level 3
°C: 	degrees Centigrade
CDC: 	Centers for Disease Control and Prevention
CLIA: 	Clinical Laboratory Improvement Amendments
CoV: 	Coronavirus
COVID-19:	Coronavirus Disease 2019
CPE:	cytopathic effect
Ct: 	Cycle Threshold
ACt:	Change/Difference in Ct Value
DMEM:	Dulbecco's Modified Eagle's Medium
EDTA	Ethylenediaminetetraacetic acid
Est.: 	Estimated
EPA: 	U.S. Environmental Protection Agency
FAM: 	6-Carboxyfluorescein
FBS:	fetal bovine serum
g: 	Gram
x g: 	centrifugation gravitational force
hr: 	hour(s)
HSRP: 	Homeland Security Research Program
IA: 	Interagency Agreement
L:	Liter
LLNL: 	Lawrence Livermore National Laboratory
[j,g: 	microgram
[xL: 	microliter
mg: 	milligram
MHV:	mouse hepatitis virus
min: 	minute(s)
mL: 	milliliter
NA:	Not Applicable
ND:	non-detect
Neg: 	negative
No.:	number
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nsp2	nonstructural protein 2
ORAU-SSC:	Oak Ridge Associated Universities-Student Service Contract
ORD:	Office of Research and Development
ORF:	open reading frame
PAPR: 	powered air purifying respirator
PBS: 	phosphate-buffered saline
PCR: 	Polymerase Chain Reaction
PFU: 	plaque forming units
Pos:	Positive
PPE:	Personal Protective Equipment
psi	pounds per square inch
RNA:	Ribonucleic acid
rpm: 	revolutions per min
RT: 	Reverse Transcriptase
RT-PCR: 	Reverse Transcriptase Polymerase Chain Reaction
RV: 	Rapid Viability
RV-RT-PCR:	Rapid Viability-Reverse Transcriptase Polymerase Chain Reaction
SARS: 	Severe Acute Respiratory Syndrome
SARS-CoV-2:	Severe Acute Respiratory Syndrome-Coronavirus-2
SD: 	standard deviation
sec:	second(s)
To: 	Time Zero (Zero Hour Incubation)
T8:	8-hr Incubation
T9:	9-hr Incubation
T12:	12-hr Incubation
Ti6:	16-hr Incubation
T24:	24-hr Incubation
Tf: 	Time (Final Number of Hours Incubation)
TBD:	To be determined
TCID50: 	50% Tissue Culture Infectious Dose
TE	Tris-EDTA
Tf: 	Time Final (Final Number of Hours Incubation)
UF:	ultrafiltration
UNG:	Uracil N-glycosylase
WHO: 	World Health Organization
X:	times concentrated
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Trademarked Products
Trademark
Holder
Location
Applied BioSystems™
Life Technologies
Carlsbad, CA
Amicon®
Millipore Corp.
Billerica, MA
ATCC®
ATCC
Manassas, VA
BD Luer-Lok™
Becton, Dickinson and Company
Canaan, CT
Biopur®, Safe-Lock™
Eppendorf
Enfield, CT
Black Hole Quencher®
Biosearch Technologies
Petaluma, CA
CRL-1586™
ATCC
Manassas, VA
Difco™
Becton, Dickinson & Co.
Franklin Lakes, NJ
Dynamag™
Life Technologies
Carlsbad, CA
EnviroMax®, Puritan™
Puritan
Guilford, ME
Excel®
Microsoft® Corporation
Redmond, WA
Invitrogen®
Life Technologies
Carlsbad, CA
Life Technologies™
Life Technologies
Carlsbad, CA
MagneSil®, MagnaBot®,
Promega™, RNasin®
Promega
Madison, WI
Millipore®, Milli-Q™
Millipore Corp.
Billerica, MA
Parafilm®
Bemis Company, Inc.
Neenah, WI
Platinum™ Taq DNA
Polymerase
Invitrogen
Carlsbad, CA
Prime Script™
Takara Bio
Mountain View, CA
Superscript™
Invitrogen
Carlsbad, CA
TaqMan®
Life Technologies
Carlsbad, CA
ToughMix®
QuantaBio
Beverly, MA
Tyvek®
Dupont
Wilmington, DE
Ziploc®
S.C. Johnson & Son
Racine, WI

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Acknowledgments
Research Team
Dr. Sanjiv Shah
(EPA Technical Lead)
Dr. Staci Kane
(Principal Investigator)
Dr. Maher El sheikh
and Teneile Alfaro
Project Team
Dr. Emily Snyder
Dr. Worth Calfee
Mr. Lance Brooks
Ms. Kathy Nickel
Ms. Sophie Manaster
Dr. Nichole Brinkman
Dr. Tonya Nichols
Dr. Christine Tomlinson
Ms. Laura Rose
Technical Reviewers
Dr. Alan Lindquist
Dr. Brian McMinn
[U.S. EPA Office of Research and Development (ORD), Center for
Environmental Solutions & Emergency Response (CESER]),
Homeland Security & Materials Management Division (HSMMD)
[U.S. Department of Energy (DOE), Lawrence Livermore National
Laboratory (LLNL)]
(U.S. EPA ORD, CESER, HSMMD)
(U.S. EPA ORD, CESER, HSMMD)
(U.S. EPA ORD, CESER, HSMMD)
(U.S. EPA ORD, CESER, HSMMD)
(U.S. EPA ORD, CESER, HSMMD, ORAU-SSC [Oak Ridge
Associated Universities-Student Service Contract])
(U.S. EPA ORD, CESER, Water Infrastructure Division)
(U.S. EPA ORD, CESER)
(U.S. EPA Office of Land and Emergency Management - Office of
Emergency Management - Consequence Management Advisory
Division)
(Centers for Disease Control and Prevention - CDC)
(U.S. EPA ORD, CESER, Water Infrastructure Division)
(U.S. EPA ORD, Center for Environmental Measurement and
Modeling, Watershed & Ecosystem Characterization Division)
External Peer-Reviewers
Dr. Paul Morin	(U.S. Food and Drug Administration)
Dr. Robin Holland	(U.S. Department of Agriculture)
Quality Assurance Reviewer
Ms. Ramona Sherman (U.S. EPA ORD, CESER, HSMMD)
Edit Reviewer
Ms. Marti Sinclair
(U.S. EPA. HSMMD, CESER: General Dynamics IT, EPA Contract
HHSN316201200050W)
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Additional Acknowledgements
We thank Laura Rose (CDC) for helpful discussions and help in acquiring Puritan swab material.
We acknowledge BEI Resources (NIAID, NIH) for the following materials: SARS-Related
Coronavirus 2, Isolate USA-WA1/2020, NR-52281; Quantitative Synthetic RNA from SARS-
Related Coronavirus 2, NR-52358.
We also thank Monica Borucki (LLNL) for providing the Vero E6 cell line, as well as Anne
Marie Erler and Dina Weilhammer (LLNL) for providing initial SARS-CoV-2 stocks.
Finally, we acknowledge Jeannette Yusko (LLNL) for excellent graphics support.
Cover Page Picture: "iStock, MatoomMi; iStock, leremy; iStock, Vectorios2016"
xiii

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Executive Summary
U.S. Environmental Protection Agency (EPA) scientists have expanded their research horizons to
respond to the public health emergency caused by the pandemic, Coronavirus Disease 2019 (COVID-
19). Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent of this
pandemic. Several research projects are underway to pave the way for better understanding and
reducing the risk of exposure to SARS-CoV-2. This research will help states and territories, tribes, and
local governments—including public health agencies—and guide business owners, homeowners, and
others in reducing the risk of exposure, which in turn will save lives.
According to the U.S. Centers for Disease Control and Prevention (CDC) as well as the World Health
Organization (WHO), transmission of SARS-CoV-2 via surfaces is not thought to be the main way the
virus spreads, however, one may get infected by touching virus-contaminated surfaces and then touching
one's mouth, nose, or eyes. Many studies have been published on SARS-CoV-2 surface contamination,
stability on different surfaces (from few hours to days), and potential indirect transmission via surfaces.
However, surface transmission of SARS-CoV-2 is not well understood, and its overall importance in
COVID-19 transmission is not well known. In part this is because the majority of studies on SARS-
CoV-2 stability on surfaces have been conducted using the reverse transcriptase polymerase chain
reaction (RT-PCR) analytical method to detect the viral RNA rather than infectious virus. Only a
limited number of studies have used a traditional cell-culture-based method that can detect infectious
SARS-CoV-2 on surfaces. Though this is considered a gold-standard method, it is laborious and takes
several days for results. Due to this limitation, it is difficult to quickly assess SARS-CoV-2 survival on
real-world surfaces and understand surface transmission. It also seriously impacts environmental
epidemiology investigations and transmission studies, where timely knowledge of the presence of
infectious virus on a surface is critical. A rapid, dependable and sensitive analytical method for
detecting infectious SARS-CoV-2 on environmental surface samples is needed to support these
investigations and aid environmental surveillance in healthcare and non-healthcare facilities, within a
community. To address this important knowledge gap, the Homeland Security Research Program
(HSRP), in collaboration with the Department of Energy's Lawrence Livermore National Laboratory
(LLNL), expeditiously developed a Rapid Viability-Reverse Transcriptase PCR (RV-RT-PCR) method
that will allow detection of infectious SARS-CoV-2 in hours, rather than several days typical of the
currently used cell-culture-based methods.
The SARS-CoV-2 RV-RT-PCR method was developed following the principle of the Rapid Viability-
PCR (RV-PCR) methods developed for detection of high-priority bacterial biological
warfare/bioterrorism agents in environmental samples under the EPA and LLNL collaboration during
2010-2019. Basically, the RV-RT-PCR method integrates cell-culture based enrichment of the virus in a
sample with virus-gene-specific RT-PCR-based molecular analysis. RT-PCR analysis of SARS-CoV-2
RNA is conducted both before and after the enrichment of the virus in cell-culture to determine the cycle
threshold (Ct) difference (ACt). An algorithm based on ACt > 6 representing ~ 2-log or more increase
in SARS-CoV-2 RNA following enrichment determines the presence of infectious virus in the sample.
An additional feature that contributes to the rapidity of this method is that the post-enrichment RT-PCR
analysis is performed while the virus is replicating in the host cells and not after complete cell lysis,
resulting in detection of the virus in hours rather than days usually taken by current cell-culture based
methods.
To efficiently establish the RV-RT-PCR method logistics, initial research work was conducted using the
murine coronavirus mouse hepatitis virus (MHV), which is closely related to SARS-CoV-2 in the
Betacoronavirus genus and can be handled under Biosafety Level (BSL)-2 laboratory conditions. The
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MHV host cell line used was 17C1-1. This work quickly determined the feasibility of using fewer 96-
well cell-culture plates instead of several large plates or flasks for the RV-RT-PCR method, allowed
selection of RNA extraction and RT-PCR kits, and established a sample processing procedure and
effective sample (virus) concentration procedure using a centrifugal ultrafiltration device. The resultant
MHV RV-RT-PCR method afforded detection of a low number of virions using a 4-hr virus-host cells
infection period and 8-hr post-infection incubation period for a total time-to-results of about 18 hours.
This method was evaluated for its performance with virus-spiked swab swatches and the method
sensitivity of detection was confirmed. Since actual swabs were not available due to their high demand
for clinical sample analysis globally, swab swatches made of the same material were used for method
development. The MHV RV-RT-PCR method will be useful in any study on surface sampling and
disinfection/inactivation, the results of which could be applied to SARS-CoV-2. An interim results
report on this research was published in September 2020
(https://www.epa.gov/healthresearch/development-rapid-viabilitv-reverse-transcriptase-pcr-rv-rtpcr-
method-detection).
Building on the MHV RV-RT-PCR method, the proof-of-concept SARS-CoV-2 RV-RT-PCR method
was developed under BSL-3 conditions using a Vero E6 host cell-line. The method parameters and
conditions were determined first using virus suspension. Multiple virus infection and post-infection
incubation periods were evaluated for a good sensitivity of detection. Sample processing procedures,
including centrifugal ultrafiltration for virus concentration developed for the MHV RV-RT-PCR method
were effectively adapted for SARS-CoV-2. Using selected parameters and conditions, the method was
tested with swab swatches spiked with SARS-CoV-2. Detection sensitivity of <50 SARS-CoV-2 virions
was achieved with just 2-hour infection and 9-hour post-infection incubation periods using spiked swab
swatches, for a total time-to-results of 17 hours. Finally, using the optimized conditions, the method
was evaluated with actual swabs that were spiked with SARS-CoV-2. The results showed that <50
SARS-CoV-2 virions can be consistently detected from swabs in a total time-to-results of 17 hours
compared to several days for traditional cell-culture-based methods.
There are many advantages of the SARS-CoV-2 RV-RT-PCR method developed in this project.
Importantly, it can reduce the time-to-results from days to hours and support timely understanding of
surface transmission of this virus. Further, the method can support epidemiology investigations and
environmental surveillance in healthcare and non-healthcare facilities, and within a community.
Another feature of this method is that it uses a 96-well format for the host cell-virus-culture, resulting in
a small footprint that is highly desirable in a BSL-3 laboratory. The small footprint and reduced waste
primarily results from (a) significantly fewer multi-well plates compared to cell-culture plates required
for traditional methods, (b) lower volumes of growth medium and reagents, and (c) less solid waste from
predominant use of micropipette tips for small volumes rather than large serological pipets required for
traditional methods. The SARS-CoV-2 RV-RT-PCR can also be a model method for other viruses.
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1.0
Introduction
The U.S. Environmental Protection Agency (EPA) scientists have expanded their research horizons to
respond to the public health emergency caused by the pandemic, Coronavirus Disease 2019 (COVID-
19). Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent of this
pandemic. Several research projects are underway to pave the way for better understanding and
reducing the risk of exposure to SARS-CoV-2 (https://www.epa.gov/emergency-response-research/epa-
expands-research-covid-19-environmenf). This research will help state, territorial, tribal, and local
governments—including public health agencies—as well as guide business owners, homeowners, and
others in reducing the risk of exposure, which in turn will save lives.
The ongoing pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-
2) continues to consume many lives worldwide, and leaves severe health effects in many recovered
patients. According to the U.S. Centers for Disease Control and Prevention (CDC), contact, droplet, and
airborne are the principal transmission modes for respiratory viruses such as SARS-CoV-2 [1-2],
Contact transmission is infection spread through direct contact with an infectious person or with a
contaminated article or surface. Surfaces can get contaminated via direct contact as well as transmitted
respiratory droplets from infected persons. Along with symptomatic patients, pre-symptomatic,
asymptomatic, and post-symptomatic carriers of SARS-CoV-2 can also shed the virus [3-12], The
environment and surfaces surrounding such virus carriers can get contaminated by droplets (coughs,
sneezes, and other exhalations) and/or surface contact in healthcare and non-healthcare settings [11-20],
Although transmission of SARS-CoV-2 via surfaces is not thought to be the primary way the virus
spreads, one could get infected by touching the virus contaminated surface and then touching one's own
mouth, nose, or eyes [1-2],
Surface contamination, stability of SARS-CoV-2 on different surfaces, and potential indirect
transmission of the virus via surfaces have been extensively discussed in several reviews [12, 21-25],
However, even more than a year after the emergence of the COVID-19 pandemic, surface transmission
of SARS-CoV-2 is still not well understood, and its overall importance in the transmission is mostly
unknown. Depending on the material, type of surface, and environmental conditions used in
experimental studies, surface stability of SARS-CoV-2 virus was reported from a few hours to days and
even up to 28 days for some conditions [26], A majority of the studies for SARS-CoV-2 stability on
surfaces have been conducted using the reverse transcriptase polymerase chain reaction (RT-PCR)
analytical method to detect the viral RNA. The RT-PCR does not distinguish between active (and
potentially infectious) and inactive (noninfectious) virus presence, and therefore, SARS-CoV-2 RNA
detected from surface samples could also be from inactive virus. Only a limited number of studies used
a cell-culture-based method that can detect infectious SARS-CoV-2 on surfaces [26-30], Traditional
viral viability methods rely on determination of cytopathic effect (CPE) that occur in host cells when
viruses replicate, and use 50% tissue culture infective dose (TCID50) calculations from multiple
dilutions of the virus in a sample. Though this method is considered a gold standard, it is laborious, and
it takes several days to get surface sample analysis results due to the long incubation times necessary for
observing CPE [26-30], Due to this limitation, it is difficult to quickly assess the SARS-CoV-2 stability
period on real-world surfaces and to understand surface transmission. It also seriously impacts
environmental epidemiology investigations and transmission studies, where timely knowledge of the
presence of infectious virus on a surface is critical. A rapid, dependable, and accurate analytical method
for detecting infectious SARS-CoV-2 in environmental surface samples (e.g., swabs) is needed to
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support understanding surface transmission of this virus. The ability to rapidly detect infectious SARS-
CoV-2 would also be valuable for epidemiology investigations and environmental surveillance in
healthcare facilities, within a facility (e.g., prison, nursing home), between facilities, and within a
community. Here, we report development of a Rapid Viability-Reverse Transcriptase PCR (RV-RT-
PCR) method that will allow detection of infectious SARS-CoV-2 in hours, rather than several days
typical of the currently used cell-culture-based methods. The method was developed in collaboration
with the Department of Energy's Lawrence Livermore National Laboratory (LLNL).
The SARS-CoV-2 RV-RT-PCR method was developed following the principle of the Rapid Viability-
PCR (RV-PCR) methods for detection of high-priority bacterial biothreat agents in environmental
samples, developed under the EPA and LLNL collaboration from 2010-2019 [31-33], Briefly, the RV-
RT-PCR method integrates cell-culture based enrichment of the virus in a sample with virus-gene-
specific RT-PCR-based molecular analysis. Using a similar cell-culture enrichment approach, the
integrated cell culture-PCR (ICC-PCR) methods were developed for other viruses [34-36], Zou et al.
[37] also combined cell-culture and RT-PCR to detect infectious SARS-CoV-2 from surfaces after 7
days incubation. The RV-RT-PCR method developed in this research effort requires RT-PCR analysis
before and after the cell-culture-virus (sample) incubation to determine the cycle threshold (Ct)
difference (ACt). An algorithm based on ACt > 6 representing ~ 2-log or more increase in SARS-CoV-
2 RNA following enrichment determines the presence/absence of infectious virus in the sample, in this
case, in hours rather than days. An additional feature that contributes to the rapidity of this method is
that the post-enrichment RT-PCR analysis is performed while the virus is replicating in the host cells
and not after complete cell lysis.
To efficiently establish the SARS-CoV-2 RV-RT-PCR method logistics, initial research work was
conducted using the murine coronavirus mouse hepatitis virus (MHV). It is closely related to SARS-
CoV-2 in the Betacoronavirus genus, and therefore, has many structural and genetic similarities with
SARS-CoV-2 [38], Most importantly, MHV is not pathogenic to humans and it can be easily
propagated and assayed in cell culture under Biosafety Level (BSL)-2 laboratory conditions. Due to
these characteristics, it was selected as a surrogate for SARS-CoV-2 [38-39], This report includes the
experimental work and results for MHV RV-RT-PCR method development. Effort with MHV included
selection of appropriate materials and procedures for cell culture, viral infection, RNA extraction, and
RT-PCR analysis. An interim results report on the initial research using MHV was published in
September 2020 (https://www.epa.gov/healthresearch/development-rapid-viabilitv-reverse-transcriptase-
pcr-rv-rtpcr-method-detection). The MHV RV-RT-PCR method can be useful in any study on surface
sampling and disinfection/inactivation for which SARS-CoV-2 cannot be used, with the results being
applicable to SARS-CoV-2.
Building on the MHV RV-RT-PCR method, the proof-of-concept SARS-CoV-2 RV-RT-PCR method
was developed in a BSL-3 laboratory using Vero E6 host cell-line. The experimental work and results
for SARS-CoV-2 RV-RT-PCR method development are described in this report. The SARS-CoV-2
RV-RT-PCR method can reduce the time-to-results from days to hours and support timely
understanding of surface transmission of this virus, as well as support epidemiology investigations and
environmental surveillance in healthcare and other facilities, and within a community. Another
advantage of this method is that it uses a 96-well format for the host cell-virus-culture resulting in a
small footprint that is highly desirable in a BSL-3 laboratory. The small footprint and reduced waste
primarily results from (a) fewer multi-well plates compared to cell-culture plates required for traditional
methods, (b) lower volumes of growth medium and reagents, and (c) less solid waste from predominant
use of micropipette tips for small volumes rather than large serological pipets required with the use of
traditional methods. The SARS-CoV-2 RV-RT-PCR can also be a model method for other viruses.
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2.0
Materials and Methods
2.1	Cell Culture and Viral Propagation
Murine coronavirus mouse hepatitis virus (MHV). The murine 17C1-1 (fibroblast) cell line was
selected for propagation of murine coronavirus mouse hepatitis virus (MHV) strain A59. MHV was
used as a BSL-2 surrogate for SARS-CoV-2. The cell line and viral stock were provided by Dr. Susan
Weiss (University of Pennsylvania).
SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2). The Vero E6 cell line (African green
monkey kidney cells; ATCC® CRL-1586™, ATCC; Manassas, VA) was selected for propagation of the
SARS-CoV-2 virus. The cell line was provided by Dr. Monica Borucki and the SARS-CoV-2 stock was
provided by Dr. Dina Weilhammer both at LLNL. The SARS-CoV-2 Isolate USA-WA1/2020 was
acquired from BEI Resources [Biodefense and Emerging Infections Research Resources Repository]
(Manassas, VA, Cat. No. NR-52281).
Cell culture conditions. The 17C1-1 and Vero E6 cells were grown and maintained in Dulbecco's
modified Eagle's medium (DMEM) (Lonza; VWR, Radnor, PA; Cat. No. 95042-512) with fetal bovine
serum (FBS; Gibco; Life Technologies, Grand Island, NY; Cat. No. 10082-147) and IX
Pen/Strep/Fungizone antibiotic (Lonza 100X, VWR; Cat. No. 12001-712) in T-75 flasks (75 cm2; TC-
treated; VWR, Secaucus, NJ; Cat. No. 75875-050). The outgrowth medium contained 10% FBS while
the maintenance medium contained 2% FBS. The cell culture media contained IX Pen/Strep/Fungizone
antibiotic for all applications, although this is often abbreviated as 2% FBS DMEM or 10% FBS DMEM
in Section 4.0 (Results and Discussion).
Both cell lines were maintained by passaging approximately twice a week. The cells sub-culture
procedure involved removing the outgrowth medium and washing the cells with 5 mL phosphate
buffered saline (PBS; VWR; Cat. No. 97062-818). Then, 0.5 mL trypsin (VWR; Cat. No. MSPP-30-
2101) was added and incubated for 3-7 minutes at 37°C in 5% CO2 in a humidified incubator. Trypsin
was then neutralized by adding 5 mL 10% FBS DMEM, using a 5-mL or 10-mL serological pipette to
pipet the medium up and down to dislodge the cells prior to transferring cells to a 50-mL conical tube
and briefly vortex-mixing. Vero E6 cells required more rigorous vortex-mixing to break up cell clumps
compared to 17C1-1 cells.
The number of cells needed for virus titration (by TCID50) was 20,000 per well in a 96-well plate with
lid (VWR; Cat. No. 10062-900), determined using a Millipore Scepter handheld cell counter (Millipore,
Billerica, MA; Cat. No. PHCC20040). For RV-RT-PCR experiments conducted over short time periods
(i.e., 8 to 24 hr), the cell count was 35,000 cells per well. The cell density was adjusted by adding fresh
outgrowth medium. Lower cell densities were used for virus titration (during several days of
incubation) to prevent overgrowth and inability to accurately determine viral infection (CPE). Vero E6
cells required agitation (rocking) while cells were settling onto 96-well plates to prevent clumping.
After viral infection, cells were cultured in the maintenance medium (DMEM with 2% FBS and IX
Pen/Strep/Fungizone antibiotic) and incubated at 37°C with 5% CO2 in a humidified incubator.
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2.2	Preparation of Viral Stocks
The following procedure was used for both MHV and SARS-CoV-2 with relevant cell lines. Two or
more T-75 flasks (75 cm2) were seeded by adding ~1 x 106 Vero E6 or 17C1-1 cells in 15-20 mL
DMEM with 10% FBS and IX Pen/Strep/Fungizone antibiotic. When cells were about 80% confluent
(after 1-2 day incubation at 37°C in 5% CO2), the medium was removed from flasks using a serological
pipette. Then, 0.5 mL of the relevant -80°C viral stock was thawed and used to infect each flask. The
viral stock source and titer was documented. The virus was spread across the entire cell monolayer by
rocking and swirling the flask by hand. The flasks were incubated for 15 min at 37°C with 5% CO2 in a
humidified incubator to allow the virus to be absorbed by the cells. Then, 15-20 mL 2% FBS DMEM
was added and the flasks were returned to the incubator. The flasks were checked daily for CPE using
an inverted microscope. When at least 75% of the cells were showing CPE, the virus was harvested by
freezing the flasks at -80°C for 10 min, and then thawing the flasks. The flasks were then rigorously
shaken from side-to-side to dislodge the monolayer and disperse the host cells. The
freezing/thawing/shaking process was repeated three times to lyse the cells and release the virions. Then
the contents of the flasks were removed using serological pipettes and pooled into one or more 50-mL
conical tubes. The tube(s) were centrifuged at 3,000-5,000 rpm at 4°C for 3-5 minutes to pellet cellular
debris. The supernatant was removed and transferred to a new 50 mL conical tube. The tube was
vortex-mixed at 1,500 rpm for 10 sec, and 0.5-mL aliquots were transferred into labeled cryovials for
storage at -80°C.
2.3	Median Tissue Culture Infectious Dose (TCID50) Analysis
Cell culture preparation for TCID50 analysis included seeding approx. 20,000 cells in each well of a 96-
well plate prior to an experiment using viral infection. When cells were above 90% confluent, 10-fold
dilution series of a viral stock were prepared in DMEM with 2% FBS and IX Pen/Strep/Fungizone. The
appropriate number of wells were infected with 0.1-mL of the viral suspension dilution in a 96-well
plate containing cell culture; for determining the MHV stock titer for suspension experiments, four
replicates were used per dilution, whereas, for remaining MHV experiments and SARS-CoV-2
experiments, twelve replicates were used per dilution to determine the viral stock titer. For TCID50
analysis to determine viral recovery efficiency from swabs or swab swatches, four replicate wells were
used per dilution and 0.1-mL maintenance medium was added to each well since recovered viral
ultrafiltration (UF)-retentates were in PBS (Sections 2.6 and 2.7). Each TCID50 plate also contained
four negative wells that were not infected. Positive controls were not included since test plates always
contained wells with known quantities of viral stock dilutions with estimated titer, however, the actual
titer of the viral stock was determined for each experiment. The plate was incubated at 37°C with 5%
CO2 in a humidified incubator for up to 10 days.
The CPE was monitored using an inverted microscope every 1-2 days. CPE resulting from cell lysis,
cell death or presence of enlarged, circular, or syncytial cells (i.e., many fused cells) were recorded. The
number of positive and negative wells was recorded and the TCID50 titer (TCID50/mL) was calculated
based on the Reed and Muench method [40], Data are expressed as TCID50 per 0.1-mL, since 0.1-mL
was used to infect cell culture wells. TCID50 analysis was conducted for every experiment in 4 or 12
replicates per 10-fold dilution to estimate the starting viral concentration. TCID50 values for stock titers
are reported along with the standard deviation in a table footnote for each experiment. This analysis was
also used to determine viral recovery efficiency after processing spiked swabs and swatches, with
TCID50 results included in a table footnote, as described below (Sections 2.6 and 2.7). The titer
expressed as plaque forming units (PFU)/0.1 mL based on the relationship PFU = 0.7 x TCID50 [41] is
also included. The standard deviation for the TCID50/0.1 mL and PFU/0.1 mL values were expressed
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as a range above and below the TCID50 or PFU per 0.1/mL, based on the Spearman and Karber
algorithm [42],
2.4 Rapid-Viability - Reverse Transcriptase PGR Method
Applying the principle of RV-PCR methods developed for bacterial biothreat agents [31-33], the SARS-
CoV-2 RV-RT-PCR method integrates cell-culture based enrichment of the virus in a sample with virus-
gene-specific RT-PCR-based molecular analysis. The RT-PCR analysis of SARS-CoV-2 RNA is
conducted both before (To) and after (Tf) the enrichment of the virus in cell-culture to determine the
cycle threshold (Ct) difference (ACt). Both To and Tf represent time in hours. An algorithm based on
ACt > 6 representing ~ 2-log or more increase in SARS-CoV-2 RNA following enrichment determines
the presence of infectious virus in the sample. Following a short post-infection incubation (hours rather
than days), RNA extraction and subsequent RT-PCR analysis is performed while the virus is replicating
in the host cells and not after complete cell lysis. The method steps are shown in Figure 1 with a
descripti on of these steps in Section 2.6 below.
Figure 1 RV-RT-PCR Method Development Flow Chart.
This general schematic shows the main protocol steps used for mock samples spiked with different titers of
SARS-CoV-2 or its surrogate, mouse hepatitis virus (MHV), for method development. Testing viral
suspension directly (not from swabs or swatches) started with the step labeled "Infect cells". The optimal
infection and incubation periods were selected based on testing (as described in Sections 2.5 and 2.6,
respectively). The 0.22 jam filtration step removes bacteria and fungi that could contaminate the cell-culture.
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2.5	RV-RT-PCR Experiments with Viral Suspension
Experiments with viral suspension were conducted to determine the optimal infection period for RV-
RT-PCR method development. The day before an experiment, the 96-well cell culture plates were
prepared as described above, with three or more wells per viral dilution and three wells for the negative
control (without virus). The same plate layout was used for the To plate and each timepoint plate (i.e.,
T8, Ti6, and T24 for MHV or T9, T12, and T24 for SARS-CoV-2). The plates were incubated at 37°C with
5% CO2 in a humidified incubator overnight, and the cell monolayer was allowed to become -100%
confluent prior to addition of virus.
Viral suspensions were prepared by thawing an aliquot (stored at -80°C) from the titered-stock and
adding 2% FBS DMEM to yield the desired viral concentrations (in 0.1 mL to be added per well) in 15-
mL conical tubes. Sufficient volume was prepared for 10-fold viral dilutions to confirm the starting
viral titer by conducting 12 TCID50 analyses (of 0.1 mL each for each 10-fold dilution) (see Section
2.3). In some cases, in addition to 10-fold dilutions, 2-fold dilutions were prepared for testing in RV-
RT-PCR experiments to evaluate method sensitivity of detection. For these cases, dilutions are
expressed as -3.3 logio and -4.3 logio, for a 2,000-fold and 20,000-fold dilution of the viral stock,
respectively. Ten-fold dilutions are referred to either as 10"2, 10"3, etc., or as -2 logio, -3 logio, etc.
Estimated viral levels added to cell culture were based on TCID50 analysis, correction for dilution, and
conversion to plaque forming units (PFU)/well as described by Leibowitz et al. [41]: PFU = TCID50 x
0.7. These PFU/well values would be multiplied by two to obtain estimated PFU/sample values since
0.1 mL was added to both To and Tf sample wells.
Next, the medium was removed from cell-culture plates by multichannel pipettor (8- or 12-channel),
taking care to not disturb the cells adhered to the bottom of the wells, and 0.1-mL of the appropriate
virus dilution was added to wells. Negative control wells had a 2% FBS DMEM aliquot (0.1-mL)
added. Plates were incubated at 37°C with 5% CO2 in a humidified incubator for 2 or 4 hr for MHV,
and 1 or 2 hr for SARS-CoV-2 with separate plates used for each combination of infection period/post-
infection incubation period. At the end of each infection period, the medium was removed from the
plate wells leaving the cell monolayer intact. Then, 0.1-mL 2% FBS DMEM was added to each well
and the plate was incubated at 37°C with 5% CO2 in a humidified incubator for the specified times. For
SARS-CoV-2 experiments, however, a wash step with 0.1 mL 2% FBS DMEM was used prior to final
medium addition. This wash step was included to remove unattached or inactive virus residuals from
inclusion in RT-PCR analysis.
Finally, for the To plate and other plates at the end of the incubation period (e.g., from 8-hr to 24-hr for
MHV or from 9-hr to 24-hr for SARS-CoV-2), the liquid volume was removed (leaving the monolayer
intact) and 0.1-mL PBS was added to each well. The plate was then stored at -80°C until RNA
extraction was conducted.
2.6	RV-RT-PCR Experiments with Swab Swatches
Experiments with swab swatches spiked with different viral levels were conducted to determine the
optimal post-infection incubation period for RV-RT-PCR method development. Swab swatches were
used since the swabs commonly used for environmental viral sampling including Puritan™ swabs (e.g.,
Cat. No. P25-88060-PF-UW-DRY) were not readily available at the project start; therefore, a sheet of
the same foam material (-4-5 mm thick) as used for environmental swabs was obtained from Puritan.
Swatches were aseptically cut using sterile scissors into -5 cm2 (2.0 x 2.5 cm) pieces, approximately
the same size as the Puritan™ swab. The swatches were then placed in individual 50-mL conical tubes.
Triplicate tubes were prepared for each viral level as well as the negative control for analysis by RV-
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RT-PCR. Viral recovery efficiency was either determined from replicate swatches or from an aliquot of
the same viral UF-retentate used for RV-RT-PCR analysis, as specified for each experiment (Section 4
Results and Discussion). Using TCID50 analysis, viral recovery from spiked swatches was determined
as follows: TCID50SWatch / TCID50stock (corrected for dilution) x 100 = percent recovery. The average %
recovery with standard deviation was determined from triplicate swab swatches processed in parallel.
For experiments, swatches were prepared by pre-wetting with 1.5-mL PBS. To the pre-wetted swatches,
0.5 mL of the appropriate viral stock dilution was spiked (expressed as either 10"2, 10"3, etc. or as -2
logio, -3 logio, etc). Then, 8-mL PBS was added for virion recovery, making the total volume 10-mL
(since swabs used for surface sampling are typically shipped in 10-mL buffer). The tubes were then
vortexed at -3,200 rpm in 15 sec bursts with 1-2 sec in between for 1 min. Next, each swatch recovered
suspension was concentrated by ultrafiltration using Amicon® Ultra-15 centrifugal filter units with 10
kDa MWCO membranes (Sigma-Aldrich, Allentown, PA; Cat. No. UFC901096). The swatch recovered
suspensions (-8-8.5 mL each) in Ultra-15 UF tubes were centrifuged at 4,000 rpm (-3200 x g) using an
Eppendorf® 5810R centrifuge with 50-mL tube rotor adapters, for -17 min at 4°C to bring the volume
down to 0.3-0.5 mL. For all tubes, the volume was brought to 0.5-mL using sterile PBS. The 0.5-mL
retentate was then filtered through a sterile 0.22-micron filter (Millipore® Cat. No. UFC30GV0S) by
centrifuging at 7,000 rpm for 1 min to remove any bacterial or fungal contaminants. The RV-RT-PCR
infection and incubation steps for swatch samples were as described above for viral suspension (Section
2.5); however, since swatch filtrates were in PBS, 0.1-mL 2% FBS DMEM was also added to each well
to provide proper medium conditions for viral infection.
A larger retentate volume of 0.5-mL was used for method development to enable testing of multiple
post-infection incubation periods (To, Ts, Ti6, and T24 for MHV and To, T9, T12, and T24for SARS-CoV-
2) from the same swatch, with 0.1-mL used for each incubation timepoint. Estimated viral levels
expressed as PFU/well are based on TCID50 analysis, corrected for the appropriate dilution, and
converted to PFU, where PFU = TCID50 x 0.7. As for viral suspension experiments, PFU/sample
would be approximated by 2 x PFU/well since 0.1 mL each is used for To and Tf aliquots from the
recovered UF-retentate.
The larger volume (0.5 mL) was also useful for TCID50 analysis of separate swatch samples (replicates)
to determine viral recovery efficiency for low viral levels. With 0.5-mL available, four aliquots of 0.1-
mL each were used undiluted (10°) for TCID50 analysis. From the remaining -0.1-mL, 0.05-mL was
used for 10-fold dilution (by adding to 0.45-mL 2% FBS DMEM) followed by 10-fold serial dilutions to
determine the TCID50 value. For some experiments as noted, 0.1-mL was available for TCID50
analysis from the same swatch sample retentate. For these swatch experiments referred to as "side-by-
side" experiments, TCID50 analysis was performed as traditionally done starting with the 10"1 dilution.
2.7 RV-RT-PCR Experiments with Swabs for SARS-CoV-2
The protocol for swatches was used for experiments with Sani-MacroSwabs (Sanigen, Anyang,
Gyeonggi-do, South Korea; www.sanigen.kr). which were made from similar materials to the Puritan
swabs. In this case, swab tubes already contained 10-mL PBS with the swab shaft attached to the tube
cap; therefore, 0.5 mL was removed prior to adding the viral dilution to the swab head and placing it
back into the tube with PBS (mimicking how a surface sample would be processed). Furthermore, due
to limited swab quantities, Sanigen swabs were only used for evaluation of the final RV-RT-PCR
protocol for SARS-CoV-2. For this evaluation of the final RV-RT-PCR method, the UF-retentate
volume was decreased from 0.5-mL to ~0.2-mL, which was split evenly between To and Tf (T9)
analyses. The TCID50 analysis (Section 2.3) was also performed on replicate swabs processed in the
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same way as those used for RV-RT-PCR analysis, and results were related to the titer of the viral stock
to determine the percent recovery as described in Section 2.6. Furthermore, percent recoveries from
higher viral levels were averaged and used to estimate a starting viral level per swatch for lower viral
levels where percent recovery was not determined directly, since these viral levels were often typically
below the TCID50 method detection limit.
2.8	RNA Extraction
For RNA extraction of viral RNA from cell culture, the MagneSil® total RNA mini isolation system
(Promega; Madison, WI; Cat. No. Z3351) was used following the manufacturer's protocol. The starting
material for extraction were individual wells on 96-well plates that contained a cell monolayer infected
with virus and covered with 0.1 mL PBS. The plates were stored at -80°C at the appropriate timepoint
(i.e., To or Tf) for RV-RT-PCR analysis. The plates were taken out of the freezer to thaw, and while still
frozen, 0.1 mL RNA Lysis Buffer from the kit was added to each well to be extracted. Once thawed, the
well contents with Lysis Buffer were pipetted up and down to mix prior to transferring to a RNase-free
2-mL snap-cap tube. Some initial testing was performed using the extraction protocol in plates (with
Promega™ MagnaBot®, magnetic plate, Cat. No. V8151); however, after discussion with Promega™
technical support, the RNA extraction protocol was adapted to 2-mL tubes for all subsequent RV-RT-
PCR experiments. A DynaMag™-2 magnetic rack (Invitrogen, Cat. No. 12321D) was used for RNA
extraction in tubes, as described for individual experiments. The Magnesil® protocol included a step
with DNAse I treatment, followed by additional wash steps, and final elution in 50 |j,L nuclease-free
water with 0.5 |j,L RNasin® Plus RNase inhibitor (Promega; Cat. No. N2615) or 0.125 [xL RNasin Plus
RNase inhibitor (Cat. No. N2615, 10,000 units). The RNasin Plus RNase inhibitor was added following
the manufacturer (Promega) guidelines to protect the RNA from exogenous RNases during storage and
handling during RT-PCR. If not analyzed immediately by RT-PCR, RNA extracts were stored at -80°C
for up to 5 days. This protocol for extraction in tubes is included with additional details in the RV-RT-
PCR Protocol (Appendix A).
2.9	MHV Reverse Transcriptase PCR Analysis
A MHV RT-qPCR assay that targets the nonstructural protein 2 gene (nsp2) and that was developed by
Case et al. [43] was used. The nucleotide sequences for the assay primers and probe are shown in Table
1. The nspl primers and probe were obtained from Biosearch Technologies (Novato, CA). Lyophilized
primers and probe were reconstituted in sterile, Tris-EDTA buffer (Fisher Scientific, Cat. No. BP2473-
500) to 100 |iM working stocks. To prepare nspl Master Mix, 10 [xM stocks were prepared in nuclease-
free water.
Table 1 Nucleotide Sequences of the Real-time RT-PCR Assay Primer/Probe Set for MHV
Assjiv
II)
(J one
T:n»el
lorwnril
Primer
Re\ erse
Primer
Probe
A in pi i con
l.en<>th (hp)
nspl
Replicase
AGAAGGTTACT
GGCAACTG
TGTCCACGGCT
AAATCAAAC
FAM-
TTCTGACAACGGCTA
CACCCAACG-BHQ
93
Nsp2 = Nonstructural protein 2, Case et al. [43],
FAM = 6-Carboxyfluorescein; bp = base pair; BHQ = Black Hole Quencher; MHV = mouse hepatitis virus.
A 5 |j,L RNA extract was used in a 25-|jL reaction using a Takara Bio One Step PrimeScript™ III RT-
PCR kit (Takara Bio; Mountain View, CA; Cat. No. RR600B). The assay conditions reported by Case
et al. [43] used 2-|jL template RNA in a 20-|jL reaction, with an qScript XLT™ One-Step RT-qPCR
8

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ToughMix® kit used on a Quantabio PCR platform. The reaction volume was increased to improve
assay sensitivity based on 10-fold dilutions of MHV RNA extracts, using both template and RT-PCR
reaction volumes (2 |j,L in 20 |j,L total vs. 5 |j,L in 25 |j,L total). Further, the conditions were adapted to
an ABI 7500 Fast real-time PCR system (Applied Biosystems Inc., Foster City, CA). The RT-PCR mix
for each nspl assay reaction is shown in Table 2. To prepare reagents for each batch of RNA extracts
from an experiment, individual component amounts were multiplied by the total number of reactions
plus 2-3 extra to account for pipetting variability.
Initially, RT-PCR conditions more closely matching those reported by Case et al. [43] were used,
however, the following conditions were appropriate for the ABI 7500 platform and provided sensitive
results: 55°C for 10 min; 95°C for 2 min; 45 cycles of 95°C for 5 sec and 60°C for 30 sec.
Table 2 Modified One Step RT-PCR Master Mix for MHV nspl Assay
Kciigcnl
Volume (ill.)
1 innl ( one. (jiM)
2X PrimeScript™ III (Takara Bio)
12.5
IX
Forward nspl primer, 10 (j,M
2.25
0.9
Reverse nspl primer, 10 |iM
2.25
0.9
Probe {nspl), 10 (j,M
0.5
0.2
Molecular Biology Grade Water
2
N/A
ROX II (5 OX)
0.5
IX
Template RNA
5
Variable
Total
25

MHV = mouse hepatitis virus.
MHV RNA extracts were also used to evaluate RT-PCR kit performance using different vendor kits
including Takara Bio One Step PrimeScript™ III RT-PCR kit and Superscript™ III Platinum™ One-
Step qRT-PCR kit (Invitrogen, Cat. No. 11745100). Ten-fold serial dilutions of the extracted MHV
RNA were prepared (as described above) in PCR-grade water and triplicate analysis were performed per
dilution. An analysis of covariance was performed on the linear regression results for both kits, with
resulting p-values for both the slope and intercept (p-values > 0.05 represented similar results).
2.10 SARS-CoV-2 Reverse Transcriptase PCR Analysis
For real-time RT-PCR analysis of SARS-CoV-2, both CDC assays N1 and N2 [43] were used. The
primers and probes were supplied by Biosearch Technologies (Hoddesdon, Herts, UK, Cat. No. KIT-
NCOV-PP1-1000). Initially, both assays were evaluated for sensitivity with quantitative synthetic RNA
standards from BEI Resources (Cat. No. NR-52358), while the N1 assay was used for method
development. For the final RV-RT-PCR protocol using swab samples, both assays were evaluated with
regard to performance (i.e., detection sensitivity with known quantities of RNA). The details on the two
assays are provided in Table 3 including gene target and genome location, with the respective sequences
for the assays listed in Table 4.
The synthetic RNA standard (BEI Resources, Cat. No. NR-52358) was supplied in RNAstable
(Biomatrica® 52201-013), as 1.05 x 108 genome equivalents/mL. The genome copy number was
determined by BioRad QX200 Droplet Digital PCR (ddPCR™) system. Ten-fold dilutions of the
standard, resulting in levels ranging from 1.5 to 1.5 x 105 viral genome copies per RT-PCR reaction,
were run with each PCR plate along with a negative control (PCR-grade water only). The synthetic
9

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standard included fragments from the ORF (open reading frame) lab, Envelope (E) and Nucleocapsid
(N) regions.
The PCR reaction conditions for N1 and N2 are shown in Table 5. As for the MHV assay, a 5-|j,L RNA
extract was used in a 25-|j,L reaction using a Takara Bio One Step PrimeScript™ III RT-PCR kit
(Takara Bio; Cat. No. RR600B). The reaction volume for the N1 and N2 assays were increased to
improve assay sensitivity and the conditions were modified for the ABI 7500 Fast Real-Time PCR
platform. The N1 primers and probe obtained from Biosearch were lyophilized together, as were the N2
primers and probe. The lyophilized reagents were reconstituted in 0.5 mL TE [Tris-EDTA] buffer to
yield working concentrations of 20 |jM primers and 5 |jM probe. Table 5 shows the reagent
composition per one reaction, and component amounts were multiplied by the total number of reactions
plus 2-3 extra to account for pipetting variability.
The RT-PCR cycling conditions were the same as those used by Lu et al. [44]: 50°C for 15 min; 95°C
for 2 min; 45 cycles of 95°C for 3 sec and 55°C for 30 sec.
Table 3 Real-time PCR Assays Used for SARS-CoV-2
Assay II)
Target
Source
Signaliiiv II)
(Jene Target /
(i on oiuo Location- -
N1
SARS-CoV-2
Biosearch
Technologies/
CDC
nCOV_N 1
Nucleocapsid,
Forward: 28303-28322
Reverse: 28374-28351
Probe: 28325-28348
N2
SARS-CoV-2
Biosearch
Technologies/
CDC
nCOV_N2
Nucleocapsid,
Forward: 29180-29199
Reverse: 29246-29228
Probe: 29204-29226
* Biosearch Technologies, Cat. No. KIT-NCOV-PP1-1000.
** Nucleotide numbering based on SARS-CoV-2 (Accession No. MN908947).
Table 4 Nucleotide Sequences of the Real-time PCR Assay Primer/Probe Sets for SARS-CoV-2
Assay
II)
Forward Primer
Re\erse I'rimor
Probe
A in p 1 i con
Si/o (hp)
nCoV
_N1
GAC CCC AAA ATC
AGC GAA AT
TCT GGT TAC TGC
CAGTTGAAT CTG
FAM-ACC CCG CAT
TAC GTT TGG TGG
ACC-BHQ
72
nCoV
_N2
TTA CAA ACA TTG
GCC GCA AA
GCG CGA CAT TCC
GAA GAA
FAM-ACA ATT TGC
CCC CAG CGC TTC
AG-BHQ
67
FAM = 6-Carboxyfluorescein; BHQ = Black Hole Quencher; bp = base pair.
10

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Table 5 One Step RT-PCR Master Mix for the SARS-CoV-2 N1 and N2 Assays
Kcsigcnl
Volume (ill.)
l-'in;il Cone.
(jiM)
2X Prime Script™ III mix
12.5
IX
Primers (20 (j,M) and Probe (5 (j,M) stock
0.625
0.5 primers
0.125 probe
Molecular Biology Grade Water
6.375
NA
ROX Dye II (5 OX)
0.5
IX
Template RNA
5
Variable
Total
25

NA = Not Applicable.
2.11 RV-RT-PCR Data Analysis and Interpretation
RV-RT-PCR algorithm for a positive virus detection result: The RT-PCR analysis of SARS-CoV-2
RNA is conducted both before (To) and after (Tf) the enrichment of the virus in cell-culture. Both To and
Tf represent time in hours. An average RT-PCR cycle threshold (Ct) was determined from triplicate
RT-PCR reactions for To and Tf RNA extracts of each sample. The average Ct of the Tf RNA extract
was subtracted from the average Ct of the To RNA extract to determine the Ct difference (ACt). If there
was no CTfor the To or Tf RNA extracts (i.e., the result was non-detect), the Ct was set to 45 (the total
number of PCR cycles used) in order to calculate a ACt value. An algorithm based on an approximately
2-log or more increase in the SARS-CoV-2 RNA following enrichment is applied such that the resultant
RT-PCR ACt > 6 between before and after cell-culture-virus incubation RT-PCR analyses determines
the presence of infectious virus in the sample. A minimum of two out of three To PCR replicates must
result in Ct values < 44 (in a 45-cycle PCR) to calculate the average Ct (for example, the average Ct for
replicate values of 42.0, 43.0, and non-detect will be 42.5).
Negative controls should not yield any measurable Ct values (i.e., a minimum of 2 of 3 RT-PCR
replicates must be non-detect). If Ct values are obtained as a result of a possible contamination or cross-
contamination, fresh RT-PCR Master Mix would be prepared and analysis would be repeated. In
addition, lab blank samples should not yield any measurable Ct values. If Ct values were observed as a
result of a possible contamination or cross-contamination, a careful interpretation of the Ct values for
the sample RNA extracts and controls must be done to determine if the data is considered valid or if the
PCR analyses must be repeated.
RV-RT-PCR analysis of sample replicates (for method development)'. Triplicate samples were used for
each test condition (i.e., viral level, infection period, and post-infection incubation period combination).
An overall average Ct value was calculated from the individual sample average Ct values. If only two
sample replicates had Ct values (to calculate an average Ct) and the third sample replicate was "non-
detect", the overall average Ct was calculated from the two samples with average Ct values. The
overall standard deviation (SD) was calculated from the following equation for 3 of 3 positive sample
replicates (with average Ct values):
Overall or joint SD = VflTn-i — 1 )si2 + (n2 — 1 )s22 + (n3 — l)s32 + (ni x \X1 — X]2) +
(n2 x [X2 - Xf) + (n3 x [X3 - X]2)]/(rh + n2 + n3 - 1)}
where nv n2, and n3 = the number of PCR analyses per sample for sample replicates 1, 2, and 3; sl3 s2,
and ,s3 = the standard deviation (SD) of the Ct values for the individual samples; Xt, X2, and X3 = the
11

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average Ct values for the individual samples; X = the overall average Ct value for the samples. The
overall SD equation was modified accordingly for only two sample replicates with Ct values.
Calculation of average A Ct and SD for triplicate samples (for method development): The average
ACt was calculated as the difference between the average Ct(To) and the average Cx(Tr), The pooled
SD for the average ACt was calculated as the square root of the following: (SD for Ct(To) values
squared plus the SD for the Ci(Tf) values squared)/2, where Tf is the post-infection incubation period.
In this effort, the RV-RT-PCR criteria for positive detection of SARS-CoV-2 was evaluated, namely,
ACt (Ct [To] - Ct [Tf]) > 6, (where f- final incubation time, hr) representing an approximately 2-1 og or
more increase in viral RNA following enrichment. Infection periods ranged from 1 to 4 hours. Testing
included 8- to 24-hr post-infection incubation periods. As mentioned, for cases where no PCR response
was obtained (non-detect results), the Ct values were set to 45 to calculate ACt (since 45 PCR cycles
were used). A ACt > 6 represented an increase in RNA concentration of approximately 2-log or more,
due to the presence of infectious virus in the original sample that propagated in cell culture during
incubation. For individual sample replicates within an experiment, the RV-RT-PCR result was
considered positive if ACt > 6 was calculated from the average of at least 2 of 3 RT-PCR replicates for
To and Tf. If a single PCR replicate was positive and the other two replicates were non-detect, the To or
Tf was considered negative or non-detect (ND) and the Ct was set to 45, in order to calculate ACt.
For triplicate sample replicates from a single viral dilution usedfor suspension experiments (Section
2.5): The average ACt and SD is not a true average ACt and SD since individual Ct values are not
correlated across the different time points. More specifically, the average ACt was calculated by taking
the average of the sample replicates for To and subtracting the average of the sample replicates for Tf.
SD represents the pooled SD, which equals the square root of the following: (SD for To values for
sample replicates squared plus the SD for the Tf values for sample replicates squared)/2, where Tf equals
T8, Tie, or T24 for MHV, and T9, T12 or T24 for SARS-CoV-2.
2.12 Biosafety
All manipulations with SARS-CoV-2 cultures were done under Biosafety Level 3 (BSL-3) conditions in
an CDC-permitted facility, including use of a certified Class II biosafety cabinet with thimble
connection and ducted exhaust, and the following personal protective equipment (PPE): Powered Air
Purifying Respirator (PAPR), Tyvek® coverall with hood and boots, disposable apron, Tyvek® sleeves,
shoe covers, and double latex or nitrile gloves. Aerosolization risk was mitigated by use of aerosol
barrier tips during pipetting and use of removable, gasketed safety cups for centrifugation that could be
loaded/unloaded in the biosafety cabinet. Secondary containment was used for 96-well plates during
incubation. Waste was subjected to two rounds of sterilization using a certified and permitted autoclave,
documented at 15 psi and 121°C for > 60 min prior to disposal.
12

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3.0
Quality Assurance and Quality Control
3.1	Laboratory Inspections
Monthly laboratory inspections were conducted by the project principal investigator to comply with
Department of Energy and Centers for Disease Control and Prevention (CDC) safety and security
policies. In addition, the LLNL responsible official and/or biosafety officer conducted annual laboratory
inspections. Inspections included the following:
Documenting laboratory cleanliness
Certifying laboratory safety equipment, including the biosafety cabinet (BSC), robotic
enclosure, and autoclave
Reviewing waste handling procedures
Taking inventory of SARS-CoV-2 stocks, sample plates, and RNA extracts (in addition, 25%
inventory conducted quarterly)
Reviewing personnel training
3.2	Calibration
The Applied BioSystems™ Inc. (ABI) 7500 Fast PCR instrument (ThermoFisher, Waltham, MA) was
calibrated and underwent preventative maintenance conducted annually. Internal instrument calibration
was conducted six months after the annual vendor calibration per the instrument software guidance.
Micropipettors were inspected and calibrated by the vendor annually; in addition, quarterly in-house
pipettor calibration was conducted gravimetrically. Balances were calibrated annually using National
Institute of Standards and Technology (NIST)-traceable standard weights. Records from these
calibration activities were documented and reviewed by the project principal investigator.
3.3	Storage Conditions
An alarm system was used for refrigerators and freezers to ensure storage conditions were within
acceptable ranges. In addition, NIST-traceable temperature-recording devices were included where
PCR reagents and frozen cell culture plates (for RNA processing) and RNA extracts were maintained.
The temperature was recorded daily to ensure the proper range was maintained. NIST-traceable
thermometers were placed in each incubator as well to provide temperature monitoring.
3.4	Spiking
Negative cell culture wells had UF-retentates from medium-spiked swabs or swatches added (in
triplicate) instead of UF-retentates from virus-spiked swabs or swatches to test for cross-contamination
for each experiment.
3.5	Real-time PCR Analysis
During the experiment, synthetic SARS-CoV-2 RNA standards were analyzed on every PCR plate,
along with the samples, as described in the Materials and Methods Section 2.10, to verify reagent quality
and instrument performance.
13

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3.6	Replication
In general, for each treatment in an experiment a minimum of three replicate samples were analyzed.
Replicate samples were spiked at the same time using the same viral dilution or viral level spiked onto
swabs or swab swatches, and processed at the same time following the same laboratory processes.
Results are presented as average Ct values (for the RV-RT-PCR method) or average TCID50 values (for
the traditional culture-based method), with corresponding standard deviation (SD).
3.7	Controls
Negative controls included in the experiments used the same matrix as the test samples with no virus
added. These controls served as a cross-contamination check and the experiment was to be repeated if
negative controls showed positive results. A negative (No-Template Control, NTC) was also included
with each PCR plate to check for PCR contamination. If the negative control showed positive PCR
results, extra care was taken to decontaminate work surfaces and prepare new reagents followed by
repeating the RT-PCR analysis.
3.8	Data Quality Objectives/Data Quality Indicators
This research effort was to develop a qualitative, RV-RT-PCR method for SARS-CoV-2 (as well as for
MHV as a BSL-2 surrogate for SARS-CoV-2). Balance, pipettor, and PCR cycler instruments were
calibrated at the following intervals: annually for the balance and cycler and quarterly for the pipettors.
Calibrations were not found to be out of range (e.g., within 0.01%). For cases where the data quality
was outside of the acceptable range (i.e., if a negative control showed 2 of 3 positive PCR results due to
potential PCR cross-contamination), the PCR analysis was repeated to ensure the expected result was
obtained. Throughout the study, negative controls showed negative results across triplicate analyses. In
addition, PCR standard curves compared between plates within an experiment were used to confirm
variability between replicate DNA standards (within 2 Ct values of the average). For individual
replicates within an experiment, the RV-RT-PCR result was considered positive when at least 2 of 3
replicates met the algorithm requirement as described in Section 2.11. In general, replicate experiments
showed consistent trends; any deviations as well as potential explanations for slight discrepancies are
included in the report.
3.9	Audit of Data Quality
This project was audited on November 18, 2020 in accordance with the Environmental Protection
Agency's Quality Assurance Program. It was determined that the data collection activities were
consistent with the project quality objectives.
14

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4.0
Results and Discussion
In this research effort, a proof-of-concept RV-RT-PCR method was developed to detect infectious
SARS-CoV-2 from swab samples. Applying the principle of RV-PCR methods developed for detection
of high-priority biothreat agents in environmental samples [31-33], the SARS-CoV-2 RV-RT-PCR
method integrates cell-culture based enrichment of the virus in a sample with virus-gene-specific RT-
PCR-based molecular analysis. The RT-PCR analysis is conducted before and after the cell-culture-
virus (sample) incubation. As described in the Materials and Methods (Section 2.11), the RT-PCR
analysis of SARS-CoV-2 RNA is conducted both before (To) and after (Tf) the enrichment of the virus
in cell-culture to determine the cycle threshold (Ct) difference (ACt). Both To and Tf represent time in
hours. An algorithm based on ACt > 6 representing ~ 2-log or more increase in SARS-CoV-2 RNA
following enrichment determines the presence of infectious virus in the sample (Figure 2). For this
method to be rapid, RT-PCR analysis was performed while the virus was replicating in the host cells and
without waiting for complete cell lysis.
Figure 2 Schematic of RT-PCR Response Curves from RV-RT-PCR Analysis of a Swab Sample.
The blue curve labeled "Time 0 response Ct (To)" represents the initial SARS-CoV-2 RT-PCR response for RNA
from virions in the sample used to infect the cell culture (immediately after the infection period). The red curve
labeled "Endpoint response Ct (Tf)" represents the SARS-CoV-2 RT-PCR response signifying viral propagation
in cell culture and the resulting increase in RNA copies detected (shown by the increased quantity of virions in the
red box relative to the blue box). Increased SARS-CoV-2 virions in cell culture result in increased SARS-CoV-2
RNA, which causes the RT-PCR response curve to shift to the left, and produces a change in cycle threshold (Ct),
or ACt, where ACt > 6 indicates the presence of infectious virus in the sample.
Since this is first reported effort to develop a SARS-CoV-2 viability testing method using such an
algorithm, it was important to first establish the method logistics using the murine coronavirus mouse
hepatitis virus (MHV). The MHV and SARS-CoV-2 are closely related viruses in the Betacoroncivirus
genus, and therefore, have many structural similarities. However, work on infectious SARS-CoV-2
must be conducted at BSL-3, while MHV can be handled under BSL-2 conditions. Therefore, the RV-
RT-PCR method logistics were established efficiently and with fewer laboratory restrictions/controls
using MHV and its host cell line, 17C1-1. The MHV RV-RT-PCR method development effort mainly
included: (a) selection of appropriate laboratory apparatus/format (e.g., 96-well plate instead of cell-
culture flasks or plates) and associated procedures for cell culture and virus infection; and (b) selection
15

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of RNA extraction and RT-PCR kits because of unavailability of the kits commonly used for SARS-
CoV-2 RT-PCR analysis due to high-demand for clinical sample analyses. Additionally, since the
swabs recommended for SARS-CoV-2 sample collection were not available, swab swatches made of the
same material were used for method development.
The research effort focused on RV-RT-PCR method development is described in this section. First, the
RV-RT-PCR method for MHV was developed and the overall method feasibility was tested. Next, the
RV-RT-PCR method for MHV was adapted for a SARS-CoV-2 proof-of-concept method, which was
evaluated for pristine conditions using viral suspension, swab swatches, and finally swabs.
4.1 Optimization of growth medium/growth conditions, and monolayer growth of the cell
line to be used as host for the surrogate virus, mouse hepatitis virus (MHV).
4.1.1	Preparation of MHV Stocks and Quantitation by TCID50 Analysis
Prior to producing a MHV stock specifically for this effort, a MHV stock from another LLNL scientist
was used; this stock was stated to contain a TCID50/mL of 106 5 although this was confirmed. For
additional experiments, MHV stocks were produced from infection of 17C1-1 cells as described in
Section 2.2. Since the growth medium and growth conditions had already been established for 17C1-1
cells and MHV by other scientists at LLNL, no further optimization was performed. As described in the
Materials and Methods section, the growth medium used was Dulbecco's modified Eagle's medium
(DMEM) with 10% fetal bovine serum (FBS) for outgrowth of cells and 2% FBS for cell maintenance
and analyses including RV-RT-PCR and TCID50. This medium also included IX Pen/Strep/Fungizone
(Section 2.1). The standard growth conditions included incubation of cells and virus in 96-well plates at
37°C with 5% C02.
The virus stock was analyzed by TCID50 (as described in Section 2.3) showing a titer of TCID50/mL =
107. The relationship between TCID50 values and plaque forming units, PFU per mL reported by
Leibowitz et al. [41] was used: TCID50/mL x 0.7 = PFU/mL. Based on this relationship, the TCID50
of 106 per 0.1 mL, or 107 per mL would have 7 x 106 PFU/mL and the TCID50 of 105 5 per 0.1 mL, or
106 5 per mL would have 2.2 x 106 PFU/mL. Both stocks (TCID50 106 and 105 5 per 0.1 mL) were used
for MHV RV-RT-PCR method development as described below.
4.1.2	Optimal Cell Seeding Density Determination
Different seeding densities of 17C1-1 cells were tested (20,000 and 50,000 cells per well) in 96-wells to
establish the time to reach confluence for TCID50 and RV-RT-PCR analyses. From this initial test, it
was determined to test higher seeding densities as well, such as 75,000 cells per well to shorten the time
for cell culture confluence, since it took two and three days to reach confluency when seeding 50,000
and 20,000 cells per well, respectively. Confluence is necessary for TCID50 analysis to accurately
distinguish CPE from cells "rounding up' prior to cell division while becoming confluent. For RV-RT-
PCR, confluent cell culture was expected to contribute to better virus infectivity resulting in better
method sensitivity of detection.
Based on additional work with the 17C1-1 cell line and MHV, the target seeding density was revised
after observing that cells that were too dense caused issues with overgrowth in the well and difficulty in
determining CPE, since cells were piling on top of one another. Table 6 shows the optimal cell seeding
densities that were established for different applications.
16

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Table 6 Seeding Densities of 17C1-1 Cells for Different Applications
Coll Culture I so
Coll Seeding Density
Comments
TCID50; for inoculation
next day
30,000
Results in 90% confluence for infection;
optimal confluence level for this cell line
TCID50; for inoculation
1.5 days later
25,000
Results in 90% confluence for infection; this
level also did not show overgrowth for the 4-
day TCID50 incubation period
RV-RT-PCR
40,000
Results in confluence without overgrowth;
higher cell levels are expected to produce
higher viral levels
TCID50 = 50% tissue culture infective dose.
The 20,000 cell level did not provide enough cells to completely cover the bottom of the well and cells
tended to migrate to the well edges leaving large gaps in the center; whereas, the 50,000 cell level
showed overgrowth and difficulty with CPE determination. The levels shown in Table 6 provided
optimal conditions for work with MHV.
Due to the need to efficiently complete the MHV work in order to initiate the SARS-CoV-2 method
development as soon as possible, work to develop suspension growth for 17C1-1 cells was not
performed. Therefore, this work was only done with the cell line used for SARS-CoV-2 propagation,
namely Vero E6 cells, as described in Section 4.3.
4.2 Optimization of incubation conditions and time intervals to determine an initial
method sensitivity of detection for MHV via RV-RT-PCR, as compared to a traditional
tissue culture infective dose (TCID50) assay, for virus from liquid suspension and
recovered from spiked swab samples.
4.2.1 RNA Extraction and RT-PCR Analysis from MHV Stock
Prior to RV-RT-PCR analysis, RNA extraction kits and RT-qPCR kits were evaluated. Although it was
initially planned to test two RNA extraction kits, namely Magnesil® Total RNA Mini-isolation System
kit (Promega) and Quick DNA/RNA Viral MagBead kit (Zymo), only the Promega™ kit was obtained
and used, with the other kit backordered without a specified delivery date. A MHV RT-qPCR assay
developed by Case, et al. [43] was selected and both published and modified RT-PCR conditions were
tested, based on discussions with the EPA Technical Lead.
For RT-PCR, Superscript III Platinum One-Step qRT-PCR (Invitrogen) and One Step PrimeScript III
RT-PCR Kit (Takara Bio) were evaluated. Initially RNA extracts from the 105 5/0.1 mL TCID50 MHV
stock were used. Using the Case et al. [43] reaction conditions of a 20-|jL reaction with 2 |j,L template,
the following RT-PCR conditions were used on a ABI 7500 Fast instrument:
• 55°C for 10 min (RT step)
95°C for 5 min (Taq DNA polymerase activation)
45 cycles of: 95°C for 30 sec (denaturation) and 60°C for 1 min (annealing/extension)
The RT-PCR results are shown in Figure 3 using dilutions of MHV RNA, comparing results from
Primescript III and Superscript III kits. Results showed > 1-log better sensitivity for the Primescript III
kit compared to the Superscript III kit, with 3.5 to 4.9 lower Ct values for Primescript across a 5-log
dilution range.
17

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The Case et al. [43] RT-PCR reaction conditions were then modified to improve assay sensitivity. In
this case, 5-|jL RNA extract was used for a 25-|jL reaction volume, and the RT conditions were as
follows:
55°C for 20 min (RT step) - extended to ensure that the RT step was complete
95°C for 2 min (Taq DNA polymerase activation) - shortened since 2 min was expected to be
sufficient and shorten the overall analysis time
45 cycles of: 95°C for 5 sec (denaturation) and 60°C for 30 sec (annealing/extension) -
shortened to preserve enzyme activity and shorten the overall analysis time
45
43
41
39
37
35
33
31
29
27
25






a
~











	J

I-,
y = -3.
R-
1367X +45.11
E = 0.9915


	a
r..t



«
K





«
k.





'*.<1


¦<
11
w
.0095X +40.802 *
K.



R2 = 0.9914

h
CT PS
CTSS
¦	Linear {CT PS)
¦	Linear {CT SS)
12	3	4
RNA Inverse Log Dilution
Figure 3 RT-PCR Results for MHV-extracted RNA with Initial Conditions.
RNA was extracted from TCID50 105 5/0.1 mL viral stock and subjected to 10-fold serial
dilution in nuclease-free water. The linear regression equations are shown.
PS = PrimeScript; SS = Superscript.
The RT-PCR results are shown in Figure 4 for these modified conditions using dilutions of MHV RNA
as described for Figure 3. The modified conditions resulted in more sensitive assay results for both RT-
PCR kits with an average 1.4 and 2.1 lower Ct values for Primescript and Superscript kits respectively,
across the range of RNA concentrations. Therefore, these conditions were used for subsequent RT-PCR
analyses for improved sensitivity, although a 10 min RT step was used since this time was considered
sufficient for cDNA conversion for the 93-base target region. Analysis of covariance of data from
Primescript and Superscript RT-PCR kits showed similar slopes (p = 0.295), suggesting assay
equivalency. However, the intercept p-values were < 0.01 (p ~ 6 x 10"13), indicating significant
differences between intercepts, and thus, assay sensitivity. Based on significantly better performance,
the Primescript RT-PCR kit was selected for use for subsequent experiments.
18

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45
43
41
39
1 37
-C
to DC
. •







y = -3.15x +43.04;
d2 _ n qq/1
I

I.






i



•






'l
L *



y = -3.3x + 39.75:
3
'¦«
~

K =
U7998T



•	CT PS
•	CT SS
	Linear (CTPS)
	Linear (CTSS)
1	2	3	4	5
RNA Inverse Log Dilution
Figure 4 RT-PCR Results for MHV-extracted RNA with Modified RT-PCR
Conditions. RNA was extracted from TCID50 105 5/0.1 mL MHV stock and subjected to
10-fold serial dilution in nuclease-free water. Linear regression equations are shown. PS =
PrimeScript; SS = Superscript.
4.2.2 RT-PCR Analysis of MHV-lnfected Cells: RNA Extraction in Tubes
It was important to ensure efficient recovery of RNA from MHV-infected cells prior to processing any
samples from RV-RT-PCR experiments in 96-well plates. For this preliminary analysis, 17C1-1 cells
were infected with MHV (1035 TCID50 per well level in growth medium) for 4 hr at 37°C in 5% CO2,
after which the medium with virus was removed and fresh medium was added, followed by incubation
at 37°C in 5% CO2 for 15.5 hr. The medium was then removed leaving the cells adhered to the plate, and
phosphate-buffered saline (PBS) was added to each well. The plate was sealed and frozen at -20°C until
RNA extraction was performed. Three replicate wells were processed in tubes (Rep 1, 2 and 3) using
the Promega™ Magnesil® RNA extraction kit, with resulting RNA (from MHV and 17C1-1 cells)
analyzed by RT-PCR using the following conditions: 55°C for 20 min; 95°C for 2 min; 45 cycles of:
95°C for 5 sec and 60°C for 30 sec.
The RT-PCR results for replicates are shown in Table 7 with the average results plotted in Figure 5. Ct
values were -17-18 for undiluted RNA from MHV-infected cells (containing RNA from both cells and
virus). The positive control was undiluted RNA extracted from the MHV stock (from TCID50 105 5/0.1
mL viral stock). Based on these results, to generate a RNA stock with a higher concentration of MHV
RNA, six additional wells were extracted from the same treatment (MHV-infected cells) using the same
Promega™ protocol. The RNA from six wells was then combined, and this RNA stock (containing
RNA from both cells and virus) was used as 10-fold serial dilutions in subsequent RT-PCR experiments.
19

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Table 7 RT-PCR Results of RNA from MHV-infected 17C1-1 Cells from 96-well Plates with
Individual Well Contents Processed in 1.5 mL Tubes
RNA
Dilution
Ct Values from RT-PCR Analysis of
Replicate Well RNA -
Processed in 1.5 mL Tubes
Avg.
SD
Rep 1
Rep 2
Rep 3
0
17.98
17.64
17.06
17.56
0.47
101
18.49
19.09
18.95
18.84
0.31
102
21.38
22.32
21.91
21.87
0.47
103
24.74
25.59
25.49
25.27
0.46
104
27.97
28.75
28.70
28.47
0.44
10 s
31.35
32.62
32.03
32.00
0.64
Neg Control
ND
ND
ND
ND
NA
Pos Control
27.30
27.13
27.02
27.15
0.14
Avg. = average of triplicate analyses; MHV = mouse hepatitis vims; NA = Not Applicable;
ND = Non-Detect (for 45 PCR cycles used); Neg = Negative; Pos = Positive; Rep =
Replicate; SD = standard deviation for triplicate analyses.
f.. |			I
y = -3.2917X + 4l[75	|
jR2 = 0.9994	^
2	3	4	5	6	7
RNA Inverse Log Dilution
Figure 5 Average RT-PCR Results for RNA from Triplicate MHV-infected Cell Culture
Wells. Dilutions from 10"1 to 10"5 were plotted as the RNA Inverse Log Dilution. Error bars
represent plus or minus one standard deviation. Linear regression results are shown. Cells were
infected with MHV at 103 5/0.1 mL TCID50 for 4 hr, followed by removal of MHV medium,
addition of fresh medium, and incubation for 15.5 hr.
4.2.3 RT-PCR Analysis of MHV-infected Cells: RNA Extraction in the Plate
Since a high-throughput sample processing and analysis method is desired, the Promega™ RNA
extraction kit performance in 96-well plates was evaluated, as compared to RNA extracted in tubes from
replicate sample wells. Triplicate wells were processed in the plate with multichannel pipettors and a
post magnet plate. Sample RNA was analyzed by RT-PCR using dilutions from 10"2 to 10"4 along with a
dilution series of the new MHV + 17C1-1 RNA stock, run in duplicate. There was an issue with the
PCR instrument returning to default conditions such that the RT Step was 55°C for only 10 sec instead
20
DD
33
31
29
27
25
23
21
19
17
11;

-------
of 10 minutes, followed by 45 cycles of: 95°C for 5 sec and 60°C for 30 sec. There was no 95°C DNA
polymerase activation (and RT inactivation) step for 2 min following the RT step. However,
comparable RT-PCR results were obtained (Table 8) showing similar RNA extraction efficiency in the
plate compared to in tubes (Table 7); for the 10"2 RNA dilution, average Ct values were 21.2 ± 0.2 and
21.9 ± 0.5, for RNA extracts in plates and tubes, respectively. These results also show that the RT step
time could be further reduced to 5 minutes or less.
The RT-PCR results from the RNA stock 10-fold serial dilutions are also plotted in Figure 6. The short
RT step was not an issue due to the small RNA target region of -93 bases. RT-PCR results for the
dilutions of RNA from plates were similar to those generated in tubes. Replicate Ct values showed little
variability (with standard deviations of 0.1-0.2).
Table 8 RT-PCR Results of RNA from MHV-infected 17C1-1 Cells
from 96-well Plates with Processing in the Plate
UNA
Dilution
( 1 Values IVoni RT-PCR Analysis of
Replicate Well R\A-
I'rocessed in the I'hile
A>«.
SI)
Rep 1
Rep 2
Rep 3
10"2
20.91
21.2
21.38
21.2
0.2
10"3
24.36
24.68
24.55
24.5
0.2
10"4
27.75
27.83
27.58
27.7
0.1
Avg. = Average (of triplicate analyses); MHV = mouse hepatitis virus; Rep = replicate;
SD = standard deviation (of triplicate analyses).
Figure 6 RT-PCR Results for RNA (MHV + 17C1-1) Dilutions.
Data are plotted as the Cycle Threshold vs. RNA Inverse Log
Dilution. Linear regression results are shown.
Based on these results, the experiment plates for RV-RT-PCR endpoint determination could be
processed using the Promega™ kit in the plate format; however, additional testing is required to
evaluate the potential for cross-contamination leading to false positive results when using either shaker
21

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plate mixing or multi-channel pipettor mixing in the plate. Alternatively, an automated RNA extraction
system may be needed for high throughput processing with plates since these systems have built in
mitigation steps to prevent cross-contamination (e.g., air gap introduced during liquid transfer by pipet,
precise positioning of pipet tips in wells, and control of aspiration speed). Regardless, RNA extraction
in tubes was used for all subsequent experiments.
4.2.4 Viral Concentration Using Amicon Ultrafiltration Devices
To enhance the RV-RT-PCR method sensitivity for swab samples, an experiment was conducted to
evaluate ultrafiltration (UF) for concentration of virions prior to RV-RT-PCR analysis. The 105 5/0.1
mL viral stock was first diluted 10,000-fold (10"4 dilution). Triplicate 12-mL aliquots from this dilution
were then loaded onto Amicon® Ultra-15 columns with 10 kDa MWCO membranes. Devices were
centrifuged at -3,200 x g for 45 min to reduce the volume to -0.24 mL. The retentate was transferred to
a sterile 0.22-micron filter insert inside a 1.5 mL collection tube so that contaminants would not be
introduced to the cell culture (i.e., UF devices are not sterile). After centrifugation at 7,000 rpm for 1
min, 0.1 mL of the filtrate was used for TCID50 analysis. The original 10"4 dilution was also analyzed
by TCID50 in triplicate. The results are shown in Table 9.
Table 9 MHV TCID50 Results with and without Concentration by 10-kDa
Ultrafiltration (UF)
Replicate
TCID50 Logio
Log
Difference
Fold
Difference
No UF
UF
1
1.25
2.5


2
1.75
3


3
1.75
2.5


Avg. (SD)
1.64 (0.29)
2.74 (0.29)
1.10
12.5
MHV= mouse hepatitis virus; UF = Ultrafiltration; TCID50 = 50% tissue culture infective
dose. Average (Avg.) and standard deviation (SD) for triplicate TCID50 analyses.
Based on volume differences, a concentration factor of 50 was possible, while the average fold increase
in TCID50 titer was 12.5 (and the logio difference was 1.10). A comparison of the lowest TCID50 value
for the "No UF" treatment and the highest TCID50 value for the "UF" treatment showed a 56-fold
difference, although with the variability between replicates, the average fold concentration was four
times lower. However, for all replicates, there was clearly increased viral titer from UF treatment.
Therefore, this clean-up/concentration step was used for subsequent experiments.
4.2.5 RV-RT-PCR Experiments with MHV Stock Dilutions
A RV-RT-PCR experiment was conducted with MHV and 17C1-1 cells to determine an optimal viral
infection period and incubation period for rapid, sensitive detection. Cells were added to 96-well plates
at 40,000 cells/well and incubated overnight at 37°C with 5% CO2 in a humidified incubator The MHV
stock (previously titered at 105 0 per 0.1 mL) was serially diluted 10-fold in cell culture medium, and the
following dilutions were added to 12 replicate wells per dilution level per timepoint (Section 2.5), using
0.1 mL per well: 10"2, 10"5, and 10"6, as well as a negative control (medium only). Each well for a given
viral level was designated as a "sample" replicate. MHV was allowed to infect for either 2 hr or 4 hr at
37°C with 5% CO2. After this time, the medium was removed and fresh maintenance medium was
added (2% FBS DMEM) to each well. At time 0 and at each post-infection incubation endpoint (8, 16,
22

-------
and 24 hr), the medium was removed and 0.1 mL phosphate-buffered saline (PBS) were added to each
well. After sealing the individual plates, they were frozen at -20°C until RNA extraction was performed.
The titer of the viral stock was also determined by TCID50 in parallel with the RV-RT-PCR experiment.
The Promega™ MagneSil® total RNA mini isolation system was used for RNA extraction of the entire
well contents (0.1 mL). The manufacturer's protocol was adapted for extraction in 1.5 or 2.0 mL tubes,
including a step with DNAse I treatment, and final elution in 50 |j,L nuclease-free water with 0.5 |j,L
RNasin Plus RNase inhibitor. After extraction, 5 |j,L RNA extract was used in a 25 |j,L reaction using a
Takara Bio One Step PrimeScript™ III RT-PCR Kit as described previously. The results for the 10"2
and 10"5 dilution levels, -700 and -0.7 PFU/well, respectively, for the 4-hr infection period are shown in
Table 10. Results for the negative control and 10"6 dilution were all non-detect (data not shown).
Table 10 RV-RT-PCR Results for MHV-infected 17C1-1 Cells with 4-hr Infection
Estimated
PFU /
Well*
Sample
Replicate
Ct by Post-Infection Incubation Period
After 4 hr Infection**
Avg. ACt (SD)***
To
T8
T16
T24
T8
T16
T24
700
1
31.6
22.7
17.3
23.6
8.2
(0.5)
14.0
(0.4)
7.7
(0.4)
2
30.9
22.9
17.2
23.6
3
31.2
23.5
17.1
23.5
Avg. (SD)
31.2 (0.4)
23.0 (0.4)
17.2 (0.1)
23.6 (0.1)
0.7
1
ND
31.4
ND
22.8
13.2
(0.4)a
21.3
(0.9)a
22.6
(0.4)
2
ND
ND
24.5
21.7
3
ND
32.1
22.8
22.8
Avg. (SD)
ND (NA)
31.8 (0.5)a
23.7 (1.2)a
22.4 (0.6)
* MHV stock titer from TCID50 analysis was 105" per 0.1 mL. PFU/Well was calculated from the TCID50/0.1 mL (corrected
for dilution) x 0.7, with 0.1 mL used per well. TCID50/0.1 mL with SD = 0.51-1.94 x 105 and PFU/0.1 mL with SD = 0.36-
1.36 x 105 (Section 2.3).
** Avg. and SD are from triplicate samples. ND is set to 45 to calculate an average Ct and ACT.
** This is not a true average ACt and SD since individual CT values are not correlated across the different time points. SD
represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for the Tf values
squared)/2, where Tf equals Ts, Ti6, or T24.
a Values are the average and SD from two positive sample replicates; one replicate was non-detect and not included in the
average and SD.
MHV = mouse hepatitis virus; PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle
Threshold; Avg. = Average; SD = Standard Deviation; ND = Non-Detect; NA = Not Applicable.
As expected, the 10"2 dilution (-700 PFU/well) showed positive results for 3 of 3 replicates with ACt >
6 for all three time periods tested. However, the ACt after 24 hr incubation was lower than that for 16-
hr incubation, possibly due to cell lysis and release of virus into the culture medium, which was
discarded. For the 10"5 dilution (-0.7 PFU/well), two of three replicates appeared positive after 8 hr
incubation, with Ct values of 31.4 and 32.1. Although the third replicate was non-detect, the average
ACt value for the positive samples was relatively high, 13.2. There were also 2 of 3 replicates positive
with 16-hr incubation with Ct values of 24.5 and 22.8, showing an even higher average ACt value of
21.3 for these two sample replicates. This viral level was slightly below the detection limit based on the
confirmed TCID50 result of the stock used to make dilutions (105 0 for 0.1 mL, the volume used to spike
each well), although a slightly higher titer was expected from previous analysis. The fact that 2 of 3
23

-------
replicates were positive (ACt > 6) at this low viral level for 8-hr and 16-hr incubation and 3 of 3 were
positive for 24-hr incubation shows the sensitivity of the method. These results may also indicate there
is some variability in the titer measurement. Subsequent experiments also included a wider range of
viral levels above the expected method sensitivity of detection.
The results for the 2-hr infection period for this experiment are shown in Table 11. The Ct values for 2-
hr infection were higher than those for 4-hr infection (Table 10) and there was less consistent positive
detection, showing that 4-hr was preferred for more sensitive RV-RT-PCR detection. Similar trends
were observed for the 10"5 dilution with 2 of 3 replicates detected at Ts, although Ct values were higher,
33.0 and 37.7. Three of three replicates were positive at T24, but one replicate had a Ct value of 43.3,
therefore this replicate was not included and the average of two sample replicates was used to calculate
ACt as noted in the table. As for the 4-hr infection period data, results for the negative control and 10"6
dilution were all non-detect and are not shown.
Table 11 RV-RT-PCR Results for MHV-infected 17C1-1 Cells with 2-hr Infection
Estimated
PFU /
Well*
Sample
Replicate
Ct by Post-Infection Incubation Period
After 2 hr Infection**
Avg. ACt (SD)***
To
t8
t16
T24
t8
t16
T24
700
1
33.1
25.5
20.5
21.5
7.6
(0.6)
12.8
(0.8)
11.5
(0.7)
2
34.1
25.8
20.0
21.7
3
33.1
26.1
21.4
22.6
Avg. (SD)
33.4 (0.6)
25.8 (0.3)
20.6 (0.7)
21.9 (0.6)
0.7
1
ND
33.0
34.6
43.3
9.6
(2.4)a
5.7
(4.6)a
23.5
(0.7)a
2
ND
37.7
43.9
22.1
3
ND
ND
ND
20.8
Avg. (SD)
ND (NA)
35.4 (3.3)a
39.3 (6.6)a
21.5 (0.9)a
* MHV stock titer from TCID50 analysis was 105 " per 0.1 mL. PFUAVell was calculated from the TCID50/0.1 mL (of the
appropriate dilution) x 0.7, with 0.1 mL used per well. TCID50/0.1 mL with SD = 0.5-1.94 x 105 andPFU/0.1 mL with SD =
0.36-1.36 x 105.
** Avg. and SD are from triplicate samples. ND is set to 45 to calculate an average Ct and ACt.
*** This is not a true average ACt and SD since individual Ct values are not correlated across the different time points. SD
represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for the Tf values
squared)/2, where Tf equals Ts, Ti6, or T24.
a Values are the average and SD from two positive sample replicates; one replicate was non-detect or >40 and not included in
the average and SD.
MHV = mouse hepatitis virus; PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle
Threshold; Avg. = Average; SD = Standard Deviation; ND = Non-Detect; NA = Not Applicable.
A second RV-RT-PCR experiment was conducted with MHV and 17C1-1 cells to confirm an optimal
viral infection period and post-infection incubation period. The 96-well plates were seeded with cells as
described above. Sample wells were infected with MHV using 10"3, 10"4, and 10"5 dilutions, along with
the negative control. For this experiment, the titer for the MHV stock was measured at 105 5 per 0.1 mL.
The results for the 4-hr infection period are shown in Table 12 (top half). In this experiment, there were
no positive sample replicates for the 10"5 dilution (~2 PFU/well) at Ts, although Ti6 showed 3 of 3
positive with Ct values of 24.0, 36.4, and 23.0 with a high ACt of-17. For the 10"4 dilution (-22
PFU/well), all 3 replicates had Ct values at Ts, while two replicates had Ct values of 40 or greater at Ts.
These Ts data were not consistent with similar samples from the first experiment, and could represent
technician error, especially since the 10"4 dilution at Ts with only 2-hr infection had Ct values of-33-35
24

-------
for all three replicate samples (Table 12, bottom half). For Ti6 and T24, all three replicates for the 10"4
dilution (-22 PFU/well) showed Ct values in the low 20's with ACt values greater than 20.
Table 12 RV-RT-PCR Results for MHV-infected 17C1-1 Cells with 4-hr and 2-hr Infection -
Replicate Experiment
Kslimated
PIT/
Well"
Sam pie
Replicate
(1 In Post-111 ledioi
After 4 lir
I	Incubation Period
II	lection-"
Avg. AC'i (SI))---
T„
Is
T„.
1 24
Is
1 u.
1 24
220
1
36.8
28.4
19.8
21.0
7.3
(1.2)
16.1
(0.9)
13.7
(1.2)
2
35.3
27.3
20.2
21.9
3
35.4
29.8
19.2
23.4
A\«.(SI»
35.8 (0.8)
28.5(1.3)
l').7 (0.5)
22.1 (1.2)
22
1
42.9
44.3
21.0
22.7
4.2
(4.0)
21.8
(1.6)
21.0
(1.4)
2
42.6
40.0
23.2
21.8
3
ND
33.6
20.9
22.9
A\«.(SI»
43.5 (1.3)"
3«>.3 (5.4)
21.7(1.3)
22.5 (0.6)
2
1
ND
ND
24.0
22.3
0.0
17.2
(5.3)
20.9
(1.7)
2
ND
ND
36.4
23.2
3
ND
ND
23.0
26.9
A\«.(SI»
NI)(NA)
NI)(NA)
27.8 (7.5)
24.1 (2.4)
Ksliniitlccl
PI I /
Well-
S;i 111 pic
Ueplic;ite
C'i hv Post-In loci ion Incuhiition Period
Al'lor 2 lir Infection
A\». ACi (SI))- - -
I",,
Is
T„.
1 24
Is
T„.
1 24
220
1
ND
32.0
25.0
21.1
11.3
(2.0)
20.6
(2.0)
22.3
(1.7)
2
42.3
34.9
21.6
21.7
3
ND
31.4
23.8
22.7
Ay... (SI))
44.1 (!.(>)'
32.8(1/))
23.5(1.7)
21.8 (0.8)
22
1
ND
35.4
27.0
22.6
10.6
(0.8)
17.8
(0.3)
20.5
(1.6)
2
ND
33.1
27.7
23.9
3
ND
34.7
26.8
27.0
Ay... (SI))
\I)(\A)
34.4(1.2)
27.2 (0.5)
24.5 (2.3)
2
1
ND
ND
ND
ND
1.2
(1.8)
-0.2
(0.3)
-0.2
(0.3)
2
44.3
ND
ND
ND
3
ND
40.8
ND
ND
Ay... (SI))
44.8 (0.4)'
43.(> (2.4) '
M) (NA)
Nl) (NA)
* MHV stock titer from TCID50 analysis was 105 5 (± 10°25) per 0.1 mL. PFU/Well was calculated from the TCID50/0.1 mL
(corrected for dilution) x 0.7, with 0.1 mL used per well. TCID50/0.1 mL with SD = 1.78-5.62 x 105 and PFU/0.1 mL with
SD = 1.24-3.94 x 105.
** Avg. and SD are from triplicate samples. ND is set to 45 to calculate an average Ct and ACt.
*** This is not a true average ACt and SD since individual Ct values are not correlated across the different time points. SD
represents the pooled SD, which equals the square root of the following: (SD for To values squared plus the SD for the Tf values
squared)/2, where Tf equals Ts, Ti6, or T24.
MHV = mouse hepatitis virus; PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle
Threshold; Avg. = Average; SD = Standard Deviation; ND = Non-Detect; NA = Not Applicable.
25

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For 2-hr infection (Table 12), fewer positive results were observed compared to 4-hr infection for the
10"5 dilution (~2 PFU/well); results for the 10"3 (-220 PFU/well) and 10"4 (-22 PFU/well) dilution were
more similar for the different infection periods. Based on increased sensitivity observed at the lowest
virus levels, a 4-hr infection period was selected for subsequent experiments with swab swatches. This
longer infection time would enable more effective viral infection to take place even for a low number of
virions recovered from samples. For both infection periods, the negative control showed non-detect
results for all replicates (data not shown).
In order to view data for the replicate experiments together, results from Table 10 and Table 12 were
combined in Table 13 for a 4-hr MHV infection period. This table includes only those viral dilutions
that yielded positive results. The summary table shows fairly consistent RV-RT-PCR results across
replicate experiments for starting TCID50 levels that differed by 0.5 log.
Table 13 Combined RV-RT-PCR Results from Replicate Experiments for MHV-infected 17C1-1
Cells with 4-hr Infection
Estimated
PFU /
Well*
Sample
Replicate
Ct by Post-Infection Incubation Period
After 4 hr Infection**
Avg. ACt (SD)***
To
T8
T16
T24
T8
T16
T24
700
1
31.6
22.7
17.3
23.6
8.2
(0.5)
14.0
(0.4)
7.6
(0.4)
2
30.9
22.9
17.2
23.6
3
31.2
23.5
17.1
23.5
Avg. (SD)
31.2 (0.4)
23.0 (0.4)
17.2 (0.1)
23.6 (0.1)
220
1
36.8
28.4
19.8
21.0
7.3
(12)
16.1
(0.9)
13.7
(12)
2
35.3
27.3
20.2
21.9
3
35.4
29.8
19.2
23.4
Avg. (SD)
35.8 (0.8)
28.5 (1.3)
19.7 (0.5)
22.1 (1.2)
22
1
42.9
44.3
21.0
22.7
4.2
(4.0)
21.8
(1.6)
21.0
(14)
2
42.6
40.0
23.2
21.8
3
ND
33.6
20.9
22.9
Avg. (SD)
43.5 (1.3)b
39.3 (5.4)
21.7 (1.3)
22.5 (0.6)
2
1
ND
ND
24.0
22.3
0.0
17.2
(5.3)
20.9
(1.7)
2
ND
ND
36.4
23.2
3
ND
ND
23.0
26.9
Avg. (SD)
ND (NA)
ND (NA)
27.8 (7.5)
24.1 (2.4)
0.7
1
ND
31.4
ND
22.8
13.2
(0.4)a
21.3
(0.9)a
22.6
(0.4)
2
ND
ND
24.5
21.7
3
ND
32.1
22.8
22.8
Avg. (SD)
ND (NA)
31.8 (0.5)a
23.7 (1.2)a
22.4 (0.6)
* MHV stock titers are from replicate experiments with TCID50 of either 105" or 105 5 per 0.1 mL. PFUAVell was calculated
from the TCID50/0.1 mL (corrected for dilution) x 0.7, with 0.1 mL used per well.
** Avg. and SD are from triplicate samples. ND is set to 45 to calculate an average Ct and ACt.
*** This is not a true average ACt and SD since individual CT values are not correlated across the different time points. SD
represents the pooled SD, which equals the square root of the following: (SD for To values squared plus the SD for the Tf
values squared)/2, where Tf equals T8, Ti6, or T24.
a Values are the average and SD from two positive sample replicates; one replicate was non-detect and not included in the
average and SD. Avg. (SD) ACt based on 2 positive replicate samples; SD represents the pooled SD as described above.
b Values are the average and SD from two positive sample replicates and one non-detect replicate with Ct set to 45.
MHV = mouse hepatitis virus; PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle
Threshold; Avg. = Average; SD = Standard Deviation; ND = Non-Detect; NA = Not Applicable.
26

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Similarly, results for viral dilutions with positive RT-PCR results using a 2-hr-infection period (from
Table 11 and Table 12) were combined in Table 14.
Table 14 Combined RV-RT-PCR Results from Replicate Experiments for MHV-infected 17C1-1
Cells with 2-hr Infection
Ksliimiled
PI 1 /
Well
S;imple
Ueplic;ile
C'i hv Post-1 nlociion Inciihiilion Period
After 2 lir In lection
A\«. AC, (SI))-- -
In
Is
In.
1 24
Is
In.
1 24
700
1
33.1
25.5
20.5
21.5
7.6
(0.6)
12.8
(0.8)
11.5
(0.7)
2
34.1
25.8
20.0
21.7
3
33 1
26 1
21 4
22 6
A\«.(SI»
33.4 (0.(>)
25.8 (0.3)
20.(> (0.7)
21/) (0.6)
220
1
ND
32.0
25.0
21.1
11.3
(2.0)
20.6
(2.0)
22.3
(1.7)
2
42.3
34.9
21.6
21.7
3
\T>
31 4
23 8
22.7
A\«.(SI»
44.1 (l.6)h
32.S (1 .*))
23.5(1.7)
21.8 (0.8)
22
1
ND
35.4
27.0
22.6
10.6
(0.8)
17.8
(0.3)
20.5
(1.6)
2
ND
33.1
27.7
23.9
3
ND
34.7
26.8
27.0
A\«.(SI»
\I)(\A)
34.4(1.2)
27.2 (0.5)
24.5 (2.3)
0.7
1
ND
33.0
34.6
43.3
9.6
(2.4)a
5.7
(4.6)
23.5
(0.7)a
2
ND
37.7
43.9
22.1
3
ND
ND
ND
20.8
A\j>. (SI))
M) (NA)
35.4 (3.3)-'
3«>.3 (6.6)-'
21.5(0/))'
* MHV stock titers are from replicate experiments with TCID50 of either 105 0 or 105 5 per 0.1 mL. PFUAVell was calculated
from the TCID50/0.1 mL (corrected for dilution) x 0.7, with 0.1 mL used per well.
** Avg. and SD are from triplicate samples. ND is set to 45 to calculate an average Ct and ACt.
*** This is not a true average ACt and SD since individual Ct values are not correlated across the different time points. SD
represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for the Tf
values squared)/2, where Tf equals Ts, Ti6, or T24.
a Values are the average and SD from two sample replicates; one replicate was non-detect or >40 and not included in the
average and SD. Avg. (SD) ACt based on 2 positive replicate samples; SD represents the pooled SD as described above.
b Values are the average and SD from one positive sample replicate and two non-detect replicates with Ct set to 45.
MHV = mouse hepatitis virus; PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle
Threshold; Avg. = Average; SD = Standard Deviation; ND = Non-Detect; NA = Not Applicable.
Together these RV-RT-PCR results for MHV suspension showed the 4-hr virus infection period
generally had more positive sample replicates than the 2-hr infection period, especially at the lowest
virus level, ~1 PFU/well. In one case for the -22 PFU/well level, the 4-hr infection period/8-hr post-
infection incubation showed only 1 of 3 positive samples, whereas the 2-hr infection period had 3 of 3
positive samples for this incubation period and virus level. This was inconsistent with the other 4-hr
infection data and could represent technician error. From these results, a 4-hr infection period was
selected for subsequent experiments with MHV-spiked swab swatches.
27

-------
4.2.6 RV-RT-PCR Analysis of MHV-spiked Swab Swatches
An experiment was conducted using swab swatches since the swabs suggested by the Center for Disease
Control and Prevention (CDC) for SARS-CoV-2 sampling were not available due to high demand and
limited supply. The recommended swabs were the same as those used for norovirus surface sampling,
and manufactured by Puritan (Cat. No. 25-88060 PF UW). Therefore, the swab foam material was
supplied by Puritan as a foam sheet and cut into swab-sized swatches. In addition, a different swab
supplier, Sanigen from South Korea, was identified by CDC for making a swab with similar materials to
that produced by Puritan. Limited Sanigen swabs (Sani-MacroSwab) were procured to use with SARS-
CoV-2 RV-RT-PCR experiments to confirm the results from experiments with the swab swatch
material. The swab swatches were cut to 2.0 x 2.5 cm (5.0 cm2), the same dimensions as the Sanigen
swab using sterile scissors in a clean biosafety cabinet.
As described in Section 2.6, the swatches were placed in a sterile 50-mL conical tube and were pre-wet
with 1.5 mL PBS. The swatches were then spiked with 0.5 mL MHV stock, which was diluted 10-fold
to different starting viral titers including 10"3, 10"4, and 10"5 dilutions, as well as a negative control (no
virus), with triplicates per viral level. In addition, viral recovery efficiency was evaluated by TCID50
analysis on replicate swatches spiked with either the 10"3 or 10"4 dilution in triplicate. Since the swabs
typically come in tubes with 10 mL buffer, 8 mL PBS was added to each swatch to bring the total
volume to 10 mL. Tubes were then vortexed at -3,200 rpm in 15 sec bursts with 1-2 sec between for 1
min. Next, the entire swatch suspension volume was recovered by pressing against the swatch with a
10-mL serological pipet and this was transferred to an Amicon® Ultra-15 (10 kDa MWCO)
ultrafiltration device. Approximately 8-8.5 mL were recovered from each swatch. The Ultra-15
devices were centrifuged at 4,000 rpm for 17 min (at 15°C) to yield retentate between 0.3 and 0.5 mL.
The retentate volume was adjusted to 0.5 mL with PBS and this was filtered through a sterile 0.22-
micron filter device (by centrifugation at 7,000 rpm for 1 min at room temperature) prior to adding 0.1
mL for each timepoint cell culture plate: To, Ts, Ti6, and T24. Filtration was performed to ensure no
bacterial or fungal contamination of the cell culture. This processing procedure was done in triplicate
for each viral level and for the negative control. In addition, 0.1 mL cell culture medium was added to
each well. The virus was allowed to infect for 4 hr at 37°C with 5% CO2 after which time, the medium
with virus was removed and 0.1 mL medium was added to each well. For the time 0 (To) plate and for
each plate at the appropriate incubation period—8, 16, or 24 hr (Tx. Ti6, and T24, respectively)—the 0.1
mL medium was removed and 0.1 mL PBS were added to each well, and the plate was sealed and frozen
at -80°C until the sample wells were extracted for RNA and analyzed by RT-PCR.
The recovery efficiency results based on TCID50 analysis are shown in Table 15, where
TCID50/swatch values were converted to PFU/swatch. Percent recovery was calculated from the
TCID50 analysis of the viral stock and from replicate swatches spiked with MHV dilution (0.5 mL per
swatch) and processed as for swatches analyzed by RV-RT-PCR. The recovery efficiencies were
estimated to range from -10 to -32% with an average of 19.8% for -1968 PFU/swatch, and values
ranged from -3.2 to -31.6% for -197 PFU/swatch with an average of 17.5%.
These recovery efficiencies include: (a) retention in the swatch itself and losses during processing steps;
(b) losses from dividing the swatch UF-retentate across 4 replicates (0.1 mL per replicate from a total
volume of 0.5 mL); and (c) losses due to inability of virions to infect cells during the 4-hr infection
period (and removal of the viral suspension used to infect cells). Viral UF-retentates are added as 0.1
mL to the cell culture well followed by addition of 0.1 mL of cell culture medium. The TCID50 method
determined an 'effective' viral recovery efficiency for infectious virions, including any inefficiencies
from the operational procedure.
28

-------
Table 15 MHV Recovery from Swab Swatch Samples
Ksliniiiled
Stsirtiii"
PI-1 /Suiilch
Swiilch
Reco\ ereil
Percent
Ksliniiiled
Ay... PI I /
Well
Repliciile
PR /Swnlch
Reco\cry

1
622
31.6

1968
2
350
17.8
-79
3
107
10.0

A\g.(SI»
-390(215)
19.8 (1 1.0)


1
35
17.8

197
2
6.2
3.2
-7
3
62
31 6

Ay... (SI))
-35(28)
17.5 (14.2)

* Values are based on dilution of the MHV titered stock (TCID50 105 75 per 0.1 mL or average 3.94 x 105 PFU/0.1 mL) with
0.5 mL used to spike the swatch (TCID50/0.5 mL x 0.7 = PFU/0.5 mL or PFU/Swatch). TCID50/0.1 mL with SD = 0.32-
1.00 x 106 and PFU/0.1 mL with SD = 2.21-7.00 x 105.
** Values are based on TCID50 analysis of swatch UF-retentates from sample processing, where TCID50/0.1 mL x 0.7 =
PFU/0.1 mL (or PFU per well). Avg. and SD are based on triplicate swatches.
*** Values are based on 0.1 mL used per well for each sample plate (T0, Tx. Ti6, and T24) from the total 0.5 mL from each
spiked swatch.
MHV = mouse hepatitis virus; PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Avg. =
Average; SD = Standard Deviation.
The RV-RT-PCR results are shown in Table 16 using the estimated PFU/well for these two viral levels.
The RV-RT-PCR data with average ACt and SD calculated is closer to a true average ACt and SD since
the recovered viral UF-retentate from the same swab was used to infect the different timepoint wells
(i.e., 0.5 mL was recovered with 0.1 mL used for each of 4 plates for To, Ts, Ti6, and T24). The negative
control values were non-detect for all three replicates for To as well as Tx and Ti6 incubation. Likewise,
results for the 10"5 dilution (~4 PFU/well) were all non-detect for all three replicates for To, Tx and Ti6
incubation. The results for the 10"3 and 10"4 dilutions (-79 PFU/well and -7 PFU/well, respectively), are
shown in Table 16. For -79 PFU/well, 3 of 3 sample replicates were detected for all of the post-
infection incubation periods with average ACt values from 10.0 to 24.6, whereas for the ~7 PFU/well
level, 2 of 3 swatches had positive results for the 8-hr and 16-hr incubation periods and 1 of 3 was
positive for the 24-hr incubation period. The RV-RT-PCR summary data for different estimated
PFU/well levels are shown in Table 17.
These data show the variability at this low viral level for different aliquots from the same swatch UF-
retentate. The variability in viral recoveries especially for the 10"4 dilution (-197 PFU/swatch or
estimated 7 PFU/well) level is consistent with 2 of 3 replicates positive for RV-RT-PCR analysis at 8-hr
and 16-hr post-infection incubation, and only 1 of 3 replicates positive for T24 incubation. Since the one
positive replicate had a high average T24 ACt value (20.9) for the same PFU level as replicates that had
non-detect results, there may have been cell culture or pipetting variability, where cell culture density
varied or some wells received more or fewer virions that others from the same stock. However, when
the final method with optimal post-infection incubation period is determined, the recovered suspension
and resulting filtered UF-retentate would only be split in half, with one half used for the To aliquot and
the other half used for the Tf aliquot for infecting cells in the respective wells. In this case, less
variability would be expected as well as more sensitive detection.
29

-------
Table 16 RV-RT-PCR Results for MHV-Spiked Swab Swatches Processed and Used to Infect
17C1-1 Cells with 4-hr Infection
list.
PI-1 /
Well*
Swiilch
Rep.
RT-IH R
Rep.
C'i hv Posl-Inloclioii 1 nciihiition Period
After 4 lir Infection1
A\«. AC, (SD)••••••
In
Is
'I'n.
1 24
Is
In.
1 24
79
1
1
ND
34.6
30.4
20.5
10.0
(0.3)
14.6
(0.1)
24.6
(0.04)
2
ND
35.1
30.3
20.4
3
ND
35.4
30.5
20.4
Avg. (SD)
ND (NA)
35.0 (0.4)
30.4 (0.1)
20.4 (0.1)
2
1
ND
28.6
29.3
21.2
15.8
(0.3)
15.1
(0.9)
23.7
(0.1)
2
ND
29.4
29.0
21.4
3
ND
29.5
31.3
21.4
Avg. (SD)
ND (NA)
29.2 (0.5)
29.9 (1.3)
21.3 (0.1)
3
1
ND
27.6
28.9
22.5
16.8
(0.3)
16.7
(0 4)
22.7
(0 1)
2
ND
28.6
27.9
22.3
3
ND
28.3
28.2
22.1
A\». (SD)
M) (NA)
2S.2 (0.5)
28.3 (0.5)
22.3 (0.2)
(hernll A\». (SI))
M)
30.8 (3.2)
29.5 (1.2)
21.4 (0.8)
14.2
(2.6)
15.5
(0.8)
23.6
(0.7)
7
1
1
ND
36.7
3U.2
ND
7.0
(0.8)
15.3
(0.4)
0.0
2
ND
38.3
29.2
ND
3
ND
39.0
29.8
ND
Avg. (SD)
ND (NA)
38.0(1.2)
29.7 (0.5)
ND (NA)
2
1
ND
31.9
ND
24.1
12.1
(0.6)
0.0
20.9
(0.1)
2
ND
33.4
ND
24.2
3
42.9
33.4
ND
24.0
A\S.(SI»
M) (NA)
32.9 (0.9)
ND (NA)
24.1 (0.1)
3
1
ND
ND
33.1
ND
0.0
12.3
(0.4)
0.0
2
ND
ND
32.1
ND
3
ND
ND
33.0
ND
A\«. (SD)
ND (NA)
ND (NA)
32.7 (0.6)
ND (NA)
()\ersil
l»os. (2
A\«. (SD)
of 3 Reps)
ND (NA)
35.5 (3.6)
31.2 (2.1)
NA
9.6
(2.3)
13.8
(1.3)
NA
* Estimated (Est.) PFUAVell is based on TCID50 analysis and viral % recovery of replicate swatches (Table 15).
** Avg. and SD are from triplicate RT-PCR analyses per swatch replicate. ND is set to 45 to calculate an average Ct and
ACt. The Overall Avg. and SD are based on three replicate swatches.
*** SD represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the Tf values squared)/2, where Tf equals Tx. Ti6, or T24.
MHV = mouse hepatitis virus; PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle
Threshold; Avg. = Average. SD = Standard Deviation; Rep. = Replicate; ND = Non-Detect; NA = Not Applicable.
30

-------
Table 17 Summary of RV-RT-PCR Results for MHV-Spiked Swab Swatches Processed and Used
to Infect 17C1-1 Cells with 4-hr Infection
Estimated
PFU /
Well*
Swatch
Replicate
Avg. Ct (SD) by Post-Infection
Incubation Period After 4-hr
Infection**
Avg. ACt (SD)***
t8
t16
t24
t8
t16
t24
79
1
35.0 (0.4)
30.4 (0.1)
20.4 (0.1)
10.0(0.3)
14.6(0.1)
24.6 (0.04)
2
29.2 (0.5)
29.9(1.3)
21.3 (0.1)
15.8 (0.3)
15.1 (0.9)
23.7(0.1)
3
28.2 (0.5)
28.3 (0.5)
22.3 (0.2)
16.8 (0.3)
16.7(0.4)
22.7(0.1)
Avg. (SD)
30.8 (3.2)
29.5 (1.2)
21.4 (0.8)
14.2 (2.6)
15.5 (0.8)
23.6 (0.7)
7
1
38.0 (1.2)
29.7(0.5)
ND (NA)
7.0 (0.8)
15.3 (0.4)
0.0
2
32.9 (0.9)
ND (NA)
24.1 (0.1)
12.1 (0.6)
0.0
20.9(0.1)
3
ND (NA)
32.7(0.6)
ND (NA)
0.0
12.3 (0.4)
0.0
Avg. (SD)
35.5 (3.6)a
31.2 (2.1)a
NA
9.6 (2.3)a
13.8 (1.3)a
NA
* Estimated PFUAVell is based on TCID50 analysis and viral % recovery of replicate swatches (Table 15).
** Avg. and SD are from triplicate RT-PCR analyses per swatch replicate. ND is set to 45 to calculate an average Ct and
ACt.
*** SD represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the Tf values squared)/2, where Tf equals T8, Ti6. or T24.
a Avg. and SD based on 2 positive replicate swatches; one replicate was non-detect and not included in the average and SD.
Avg. (SD) ACt based on 2 positive replicate swatches; SD represents the pooled SD as described above.
MHV = mouse hepatitis virus; PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle
Threshold; Avg. = Average. SD = Standard Deviation; ND = Non-Detect; NA = Not Applicable.
A replicate RV-RT-PCR experiment was conducted using swab swatches as described previously. In
this experiment, 0.5 mL of MHV diluted to 10"3, 10"4, or 10"5 relative to the stock (3.94 x 105 PFU/0.1
mL), as well as negative controls (without virus) were added to triplicate swatches. In addition, to
evaluate viral recovery efficiency, replicate swatches were spiked with the 10"3 (-1970 PFU/swatch) or
10"4 dilution (-197 PFU/swatch) in triplicate. Relating the starting stock TCID50 results corrected for
dilution with TCID50 values from swatch UF-retentates, the recovery efficiencies were estimated to
range from -10 to -56% with an average of 32.6% for the -1970 PFU/swatch; values ranged from -5.6
to -17.8% for -197 PFU/swatch with an average of 11.1% (Table 18). Due to such a low recovery
efficiency, the UF-device-based cleanup and concentration step is important to detect a low number of
virions in a sample and avoid false negative results.
The results for the 10"3 and 10"4 dilutions, or -128 PFU/well and -4 PFU/well levels, respectively, are
shown in Table 19. For -128 PFU/well, 3 of 3 swatch replicates were detected for all of the post-
infection incubation periods with overall average ACt values from 12.1 to 21.9, whereas for -4
PFU/well, 1 of 3 swatches had positive results for all three incubation periods and 2 of 3 swatches had
positive results for the 8-hr and 16-hr incubation periods. For this viral level, one sample was only
positive for the 24-hr incubation period. Average ACt values for these positive swatches were 10.2 for
To - T8, 19.5 for To - Ti6, and 24.8 for To - T24. If one determined the method sensitivity of detection
from this data, it would be between these two starting viral levels, since there was 3 of 3 swatches
detected for the 10"3 dilution (-128 PFU/well) and 2 of 3 detected for the 10"4 dilution (-4 PFU/well).
Percent recoveries were similar to those from the first experiment, although the average value for the
10"3 dilution was slightly higher, -33% compared to -20%, whereas the average value for the 10"4
31

-------
dilution were slightly lower, ~11% compared to -18%. As mentioned, virions can be lost to the swab
matrix, the ultrafiltration device, the 0.22-micron filter, and pipet tips and tubes. In addition, dividing
the sample UF-retentate across 4 replicate plates (for To, T9, T12, and T24) and any inability of recovered
virions to infect cells would affect the recovery efficiency results. The low viral recoveries for the -4
PFU/well level is consistent with 2 of 3 replicates positive for RV-RT-PCR analysis across the three
timepoints. As mentioned, the swab UF-retentate would be split in half for To and Tf analysis for each
sample for the final method, therefore efficiencies would be improved by a factor of 2 or more.
Table 18 MHV Recovery from Swab Swatch Samples - Replicate Experiment
KsliniiiU'd
Stiirliii"
I'l l /Swatch*
Swnlcli
Reco\ crcil
Percent
Kstimnlccl
A>«.
PI-1 /Well
Rcplicitle
PI I /Swnlcli
Rcco\cry

1
622
31.6

1970
2
197
10.0
-128
3
1107
56.2

Ay... (SI))
(>42 (455)
32.6(23.1)


1
35
17.8

197
2
20
10.0

3
11
5.6

A\«.(SI»
22(12)
1 I.I ((>.2)

* Values are based on dilution of the MHV titered stock (10575 per 0.1 mL or average 3.94 x 105 PFU/0.1 mL) with 0.5 mL
used to spike the swatch. TCID50/0.1 mL with SD = 0.32-1.00 x 106 and PFU/0.1 mL with SD = 2.21-7.00 x 105.
** Values are based on TCID50 analysis of swatch UF-retentate from sample processing. Avg. and SD are based on triplicate
swatches.
*** Values are based on 0.1 mL used per well for each plate (T0, Tx. Ti6, and T24) from the total 0.5 mL from each spiked and
processed swatch.
MHV = mouse hepatitis virus; PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Avg. =
Average; SD = Standard Deviation.
The negative controls were non-detect for all three replicates for all incubation periods (data not shown).
The results for the 10"5 dilution (-19.7 PFU/swatch) were non-detect for all three sample replicates (and
all three RT-PCR replicates) for To, Ti6, and T24; however, for Ts, this dilution showed 3 of 3 replicates
with average Ct values of 36.2-39.9. In addition, repeating the RT-PCR analysis also showed similar
Ct values for all three sample replicates and RT-PCR replicates per sample. The results for Tx were
reproducible and the negative controls were non-detect, showing these results were valid. Detection at
T16 and T24 would be expected for at least some of the replicates; however, more variability would be
expected at this high viral dilution (low viral concentration). In this experiment, the viral UF-retentate
was recovered from the same swab swatch and used to infect To, Ts, Ti6, and T24 for each sample. This
approach was used to quickly determine the optimal timepoint for Tfmai (Tf); however, for the final
method, the filtered UF-retentate would be split in half, with one half used To and one half for Tf to
infect cell culture wells. This would result in a lower sensitivity of detection since the entire volume
would be used for RV-RT-PCR analysis, split between To and Tf aliquots.
32

-------
Table 19 RV-RT-PCR Results for MHV-Spiked Swab Swatches Processed and Used to Infect
17C1-1 Cells with 4-hr Infection - Replicate Experiment



Ci hv I'ost-lnlcclion Incii hill ion Period
A>«.
AC, (S1)J---------
hsl.
pi- i /
Well
Swjilch
R'I'-PCR

Al'ler 4 lir In loci ion

Rep.
Replicittc
I",,
Is
1 l(.
1 24
Is
'I'ii.
t:4


1
40.5
28.5
22.6
21.3




1
2
40.8
28.5
22.3
21.7
12.1
18.3
18.9

3
40.9
28.7
22.3
22.3
(0.3)
(0.2)
(0.4)


Avg. (SD)
40.7 (0.2)
28.6 (0.4)
22.4 (0.2)
21.8 (0.5)





1
ND
31.6
21.5
19.6




2
2
43.4
31.7
21.0
18.8
11.1
21.6
23.4
128
3
42.3
31.8
21.1
19.9
(0.5)
(0.5)
(0.7)

Avg. (SD)
42.8 (0.7)
31.7 (0.1)
21.2 (0.2)
19.4 (0.6)





1
41.4
29.1
23.6
18.6




3
2
42.5
28.8
23.0
18.8
13.0
18.8
23.4

3
ND
29.0
23.0
18.5
(0.6)
(0.6)
(0.6)


Avg. (SD)
42.0 (0.8)
29.0 (0.2)
23.2 (0.4)
18.6 (0.1)




Oxernll
\\g. (SI))
41.8(1.1)
2«>.8 (1.5)
22.3 (0/))
1')/) (1.5)
12.1
(0/))
1').(»
(1.6)
21/)
(2.3)


1
ND
32.3
25.U
2U.5




1
2
ND
32.6
24.9
20.9
12.5
20.1
24.2

3
42.3
32.7
24.8
20.9
(0.2)
(0.1)
(0.1)


Avg. (SD)
ND (NA)
32.5 (0.3)
24.9 (0.1)
20.8 (0.2)





1
ND
ND
ND
19.5




2
2
ND
ND
ND
19.8
0.0
0.0
25.3
4
3
ND
ND
ND
19.8
(0.1)


Avg. (SD)
ND (NA)
ND (NA)
ND (NA)
19.7 (0.2)





1
ND
37.6
26.2
ND




3
2
ND
36.7
26.1
ND
7.9
18.9
24.8

3
ND
37.2
26.0
ND
(0.4)
(0.7)
(0.6)


Avg. (SD)
ND (NA)
37.1 (0.5)
26.1 (0.1)
ND (NA)




Oxei itll A\». (SI))
M) (NA)
34.8
(2.0)-
25.5
(0.5)-
20.3
(0.5)-
10.2
(2.5)-
l»>.5
(0.7);1
24.8
(0.6)-
* Estimated (Est.) PFUAVell is based on TCID50 analysis and viral % recovery of replicate swatches (Table 18).
** Avg. and SD are from triplicate RT-PCR analyses per swatch. ND is set to 45 to calculate an average Ct and ACt. For 1 of
3 positive RT-PCR replicates, the sample result is considered ND. The Overall Avg. and SD are based on three replicate
swatches.
*** SD represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the Tf values squared)/4, where Tf equals Ts, Ti6, or T24.
a Avg. and SD based on 2 positive replicate swatches; one replicate was non-detect and not included in the average and SD.
Avg. (SD) ACt based on 2 positive replicate swatches; SD represents the pooled SD as described above.
MHV = mouse hepatitis virus; PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle
Threshold; Avg. = Average; SD = Standard Deviation; Rep. = Replicate; ND = Non-Detect; NA = Not Applicable.
33

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The summary data with average Ct per swatch replicate and Avg. ACt values for these starting
PFU/well level are shown in Table 20. In addition, the RV-RT-PCR results for replicate swatch spiked
with low MHV levels are summarized in Table 21.
Table 20 Summary of RV-RT-PCR Results for MHV-Spiked Swab Swatches Processed and Used
to Infect 17C1-1 Cells with 4-hr Infection - Replicate Experiment
Estimated
PFU/Well*
Swatch
Replicate
Avg. Ct by Post-Infection Incubation Period
After 4 hr Infection**
Avg. ACt (SD)***
To
T8
T16
T24
T8
t16
T24
128
1
40.7 (0.2)
28.6(0.4)
22.4 (0.2)
21.8 (0.5)
12.0
(1.3)
19.5
(1.2)
21.9
(1.3)
2
42.8 (0.2)
31.7(0.1)
21.2 (0.2)
19.4 (0.6)
3
42.0 (0.8)
29.0 (0.2)
23.2 (0.4)
18.6(0.1)
Avg (SD)
41.8 (1.1)
29.8 (1.5)
22.3 (0.9)
19.9 (1.5)
4
1
ND (NA)
32.5 (0.3)
24.9 (0.1)
20.8 (0.2)
10.2
(i.or
19.5
(0-3)a
24.7
(0.2)a
2
ND (NA)
ND (NA)
ND (NA)
19.7(0.2)
3
ND (NA)
37.1 (0.5)
26.1 (0.1)
ND (NA)
Avg (SD)
ND (NA)
34.8 (2.0)a
25.5 (0.5)a
20.3 (0.5)a
* Estimated PFUAVell is based on TCID50 analysis and viral % recovery of replicate swatches (Table 18).
** Avg. and SD are from triplicate RT-PCR analyses per swatch. ND is set to 45 to calculate an average Ct and ACT.
*** SD represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the Tf values squared)/4, where Tf equals Tx. Ti6, or T24.
a Avg. and SD are based on 2 positive replicate swatches; one replicate was non-detect and not included in the average and
SD or pooled SD.
MHV = mouse hepatitis virus; PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle
Threshold; Avg. = Average; SD = Standard Deviation; ND = Non-Detect; NA = Not Applicable.
Table 21 Combined RV-RT-PCR Results from Replicate Experiments for MHV-Spiked Swab
Swatches Processed and Used to Infect 17C1-1 Cells with 4-hr Infection and 8-hr Post-Infection
Incubation
Estimated PFU/
Well*
Avg. T8ACt (SD)**
for 4 hr Infection
Positive Swatch Replicate
Results
128
9.5 (1.8)
3 of 3
79
14.2 (2.6)
3 of 3
7
9.6 (2.3)a
2 of 3
4
9.6 (3.1)a
2 of 3
* Estimated PFUAVell is based on TCID50 analysis and viral % recovery of replicate swatches (Tables 15 and 18),
where PFUAVell = TCID50/well x 0.7.
** SD represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD
for the Ts values squared)/2.
a Avg. and SD are based on 2 positive replicate swatches; one replicate was non-detect and not included in the average
and SD.
MHV = mouse hepatitis virus; PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct =
Cycle Threshold; Avg. = Average; SD = Standard Deviation.
The MHV RV-RT-PCR experiments laid the foundation for development of the SARS-CoV-2 RV-RT-
PCR method, specifically as follows: (a) the method can be performed in 96-well plate instead of cell-
culture flasks or plates by establishing optimum host cells seeding density; (b) the Promega™ RNA
extraction kit and the Takara Bio RT-PCR kit could be effectively used; (c) the Amicon® Ultra-15
34

-------
centrifugal filter device can be effectively used for sample (virus) cleanup/concentration; (d) method
sensitivity of detection achieved with the virus suspension can be maintained when swab swatches
spiked with virus are used; and (e) the RV-RT-PCR method can yield analytical results in hours rather
than several days taken by the traditional cell-culture-based method. In general, the results indicated
that a low number of MHV virions could be detected in about 18 hours, which included 2-hr front-end
sample processing, a 4-hr infection period, a 8-hr post-infection incubation period, and a 4-hr back-end
RNA extraction and RT-PCR analysis (i.e., 18 hours total time-to-results).
4.3 Evaluation and optimization of growth conditions in a multi-well plate format, and
monolayer and suspension growth of the host cell line for the SARS-CoV-2 virus.
4.3.1	Establishing Optimal Vero E6 Cell Preparation Procedures for SARS-CoV-2
Propagation
As described in the Materials and Methods section, the growth medium used for Vero E6 cell culture
and SARS-CoV-2 propagation was Dulbecco's modified Eagle's medium (DMEM) with 10% fetal
bovine serum (FBS) for outgrowth of cells and 2% FBS for cell maintenance. The standard growth
conditions included incubation of cells and virus in 96-well plates at 37°C with 5% CO2.
The Vero E6 cells used in this effort were obtained from another LLNL scientist, from the same stock
being used for SARS-CoV-2 propagation on other LLNL projects. To prepare a 96-well culture plate
with Vero E6 cells, the culture medium was removed from the starting flask (T-75), then cells were
washed with PBS, and subsequently treated with trypsin, to release the cells from the surface. Next the
trypsin was neutralized and 5-mL culture medium (DMEM with 10% FBS, Section 2.1) was added.
Cell density was determined by using a Scepter automated cell counter. The cell density was adjusted to
400,000 cells/mL with 0.1 mL added to appropriate wells on a 96-well culture plate (40,000 cells/well).
After overnight growth in the plates, cell clumping was noted that could interfere with RV-RT-PCR
analysis. The Vero E6 cells proved to be more difficult to dislodge from the surface and disperse than
the 17C1-1 cells used for MHV; therefore, the amount of trypsin was increased from 0.5 mL to 1.0 mL
per flask, and the trypsin treatment time was increased from 1 min to 5 min. After trypsin treatment, the
cells were vortexed with a tube vortexer at maximum speed for 30 sec to ensure there was no cell
clumping. In addition, the seeding density was decreased to 35,000 cells/well for RV-RT-PCR analysis.
For TCID50 analysis, the optimal seeding density was determined to be 25,000 cells/well to establish a
monolayer (and prevent overgrowth), since these plates are incubated in a humidified incubator for up to
10 days. This seeding density yielded -90-100% confluency by the next day.
In addition, the plate required oscillating after one hr of seeding to prevent cell clumping. These
procedure modifications routinely produced Vero E6 cells at greater than 90% confluency in wells the
next day, without any clumping. While some dead cells were observed to be floating in the culture
wells, these were easily removed upon changing the medium, prior to infection with viral dilutions.
4.3.2	Evaluation of Suspension Growth of Vero E6 Cell Cultures
To assess the feasibility of using suspended cells for RV-RT-PCR analysis and thereby enable taking
aliquots from the same sample for To and Tf analysis, experiments were initiated using different starting
Vero E6 cell densities in 6-well untreated cell culture plates. It was determined that up to 4 mL medium
could be used with orbital shaking at 140 rpm to potentially prevent cells from adhering to the plate.
The first tests used cell densities ranging from 10,000 to 50,000 per well; however, the cells were not
maintained in the liquid phase within 7 days, likely due to cell death/lysis. In a subsequent experiment,
the cell density was increased to 80,000 to 280,000 cells per well. For these tests, wells with lower cell
35

-------
numbers had visibly lower cell numbers within 3 days and also showed a monolayer forming on the
bottom of the well and some clumping of cells in suspension. The wells with higher starting cell
numbers also showed decreased cell density and monolayer formation and larger clumps of suspended
cells. After 7 days, individually suspended cells for all starting levels except the 280,000 level were no
longer evident. All wells had a larger monolayer attached to the bottom in addition to clumps of cells in
suspension. The cell clumps were shaped as a layer of cells, not rounded aggregates.
A 2-mL aliquot of the remaining medium in wells was then transferred to a new 6-well plate and
additional 2-mL fresh medium was added to each well. After 2 days incubation with shaking, an
attached monolayer started forming. Suspension pieces of flat clumps of cells also increased in all
wells. The well corresponding to the highest number of cells initially (280,000) had the most small cell
clumps in addition to the larger clumps that were flat in shape. All other wells had larger clumps of
aggregated cells in suspension. These initial findings showed it was difficult to obtain consistent
cultures of suspended Vero E6 cells. Additional studies using large cell densities with transferring only
suspended cells after every 2-3 days by mixing 1:1 with fresh medium also failed to adapt cells to
suspended growth conditions. Therefore, only Vero E6 cells in monolayer, adhered to 96-well plates,
were used for SARS-CoV-2 RV-RT-PCR experiments.
4.4 Optimization of virus infection period and post-infection incubation period to
determine an initial method sensitivity of detection of SARS-CoV-2 via RV-RT-PCR, for
virus from liquid suspension and recovered from spiked swab samples.
4.4.1 Evaluation of SARS-CoV-2 RT-PCR Assays
For real-time RT-PCR analysis of SARS-CoV-2, signatures and respective PCR assays were developed
by the CDC [43] and made available for both clinical and environmental sample analysis. Primers and
probes for SARS-CoV-2 assays were obtained from Biosearch Technologies, Inc. from production lots
that were approved by the CDC. Table 22 shows the details on the assays, including gene target regions,
and primer and probe sequences used for RV-RT-PCR method development.
Table 22 CDC Real-time PCR Assays for SARS-CoV-2 Detection
Assay
ID
Signature
ID
Gene Target*/
Genome Location* /
Amplicon Size (bp)
Forward
Primer
Reverse
Primer
Probe
N1
nCOV_Nl
Nucleocapsid /
Forward: 28303-28322
Reverse: 28374-28351
Probe: 28325-28348 / 72 bp
GAC CCC
AAA ATC
AGC GAA
AT
TCT GGT
TAC TGC
CAG TTG
AAT CTG
FAM-ACC CCG
CAT TAC GTT TGG
TGG ACC-BHQ
N2
nCOV_N2
Nucleocapsid /
Forward: 29180-29199
Reverse: 29246-29228
Probe: 29204-29226 / 67 bp
TTA CAA
ACA TTG
GCC GCA
AA
GCG CGA
CAT TCC
GAA GAA
FAM-ACA ATT
TGC CCC CAG
CGC TTC AG-BHQ
* Nucleotide numbering based on SARS-CoV-2 (Accession No. MN908947); bp = base pair.
FAM = 6-Carboxyfluorescein; BHQ = Black Hole Quencher.
The PCR conditions specified by Lu et al. [44] were as followed with changes specified in Table 23.
The One-Step RT-PCR kit used by CDC was not available to non-CLIA (Clinical Laboratory
Improvement Amendments) laboratories processing environmental samples. The Takara Bio
Primescript III One Step RT-PCR kit was down-selected for the MHV RT-PCR assay (since it was ~1-
36

-------
log more sensitive than the Invitrogen Superscript III kit); therefore, this kit was also used for SARS-
CoV-2 assays. The Takara Bio kit does not include Uracil N-glycosylase (UNG), so this step was
removed from that reported by the CDC. UNG is an enzyme used to eliminate carryover PCR products
in Real-Time, quantitative PCR. The PCR cycling conditions were as follows: 50°C for 15 min for RT
step; 95°C for 2 min to inactivate the RT enzyme and activate the Taq DNA polymerase enzyme; 45
cycles of 95°C for 3 sec and 55°C for 30 sec.
Table 23 One Step RT-PCR Mix for SARS-CoV-2 N1 and N2 Assays Modified from CDC
Uc«i«icnt
Volume
(.ill)
1 iiiiil C onceiili itlioii
(jiM)
Chsingc from
l.ii el ill. (44)
2X One Slop h'imcScnpl1 M 111 R l -
qPCR Mix (Cat. No. RR600B)
12.5
IX
4X \lasLcr Mix
(5 pL)*
Primers (20 (j,M) and Probe (5 |iM)
stock (rehydrated from lyophilized)
0.625
0.5 primers
0.125 probe
Primers and probe separate
stocks (0.5 |_iL each)
Molecular Biology Grade Water
6.375
N/A
8.5 pL
ROX Dye II (5 OX)
0.5
IX
Included in Master Mix
Template RNA
5
Variable
No change
TOTAL
25

20 pL total
* TaqPath 1-Step RT-qPCR Master Mix, CG (Thermo Fisher Scientific). This reagent/kit is only available to Clinical
Laboratory Improvement Amendments (CLIA)-approved laboratories analyzing clinical samples.
Results for RT-PCR analysis of synthetic RNA from SARS-CoV-2 (BEI Resources) using N1 and N2
assays is shown in Table 24 and Table 25, respectively. The data are also plotted for both assays in
Figure 7. The RNA quantities in the table are based on 1.05 x 108 genome equivalents/mL for the lot
number supplied by BEI. The data showed good assay sensitivity for both N1 and N2 with consistent
detection of 1-2 RNA copies, showing that the changes outlined in Table 23 to adapt the assay to the
Takara Bio RT-PCR reagents and 25-|iL volume did not negatively affect assay performance. Based on
comparable performance between assays (two-tailed, paired t-test, p > 0.05; analysis of covariance for
slopes, p -0.98), the N1 assay was used for method development, and both assays were used for final
RV-RT-PCR method evaluation.
Table 24 RT-PCR Results for SARS-CoV-2 N1 Assay with Synthetic RNA
UNA Stock
10-1-old
Dilution
UNA
(.CIlOlllC
Kquh iilonts
A\«. ( i (SI))
()\enill A\«.(SD)
Rep 1
Rep 2
101
5.25 x 104
21.3 (0.03)
21.2 (0.02)
21.3 (0.04)
102
5.25 x 103
24.8 (0.02)
24.8 (0.01)
24.8 (0.02)
103
5.25 x 102
28.4 (0.06)
28.4 (0.08)
28.4 (0.05)
104
5.25 x 101
32.2 (0.1)
31.9(0.1)
32.0 (0.2)
10 s
5.25 x 10°
35.7(0.6)
35.7(0.5)
35.7 (0.4)
106
5.25 x 10"1
38.9(1.0)
38.9(0.9)
38.9 (0.7)
Neg Control
0
ND
ND
ND
* BEI Resources, Cat. No. NR-52358 (1.05 x 108 genome equivalents per mL).
** Automated cycle threshold and baseline setting used. Rep = Replicate; Neg = Negative.
Average (Avg.) of triplicate RT-PCR analyses for each 10-fold dilution for replicate experiments (Rep 1
and Rep 2); SD = standard deviation (from triplicate analyses); ND = Non-Detect.
37

-------
Table 25 RT-PCR Results for SARS-CoV-2 N2 Assay with Synthetic RNA
RNA Stock
10-Fold
Dilution
RNA
Genome
Equivalents*
Avg. CT (SD)**
Overall Avg. (SD)
Rep 1
Rep 2
101
5.25 x 104
21.0 (0.02)
20.8 (0.03)
20.9 (0.09)
102
5.25 x 103
24.4 (0.05)
24.4 (0.1)
24.4 (0.06)
103
5.25 x 102
28.0 (0.09)
27.9 (0.05)
28.0(0.07)
104
5.25 x 101
31.9 (0.07)
31.7(0.2)
31.8 (0.1)
10 s
5.25 x 10°
35.4 (0.4)
34.8 (0.3)
35.1 (0.3)
106
5.25 x 10"1
38.9(0.6)
38.9(0.7)
38.9 (0.5)
Neg Control
0
ND
ND
ND
* BEI Resources, Cat. No. NR-52358 (1.05 x 108 genome equivalents per inL).
** Automated baseline setting used. The cycle threshold was set manually using the same threshold as
that used for the N1 assay. Rep = Replicate; Neg = Negative. Average (Avg.) of triplicate RT-PCR
analyses for each 10-fold dilution for replicate experiments (Rep 1 and Rep 2); SD = standard deviation
(from triplicate analyses); ND = Non-Detect.
2
o
>
u
35
30
25
20
-t5-
"""V,
®l

N1
y= -3.5601X +38.082
R2 = 0.9997

• N1CT
A N2CT
N2
y=-3.5903x +37.815
R2 = 0.9997
12	3
Log RNA Genome Equivalents
Figure 7 Average RT-PCR Results Using N1 and N2 Assays with Synthetic SARS-CoV-2
RNA. Data points represent average values from duplicate dilutions series with triplicate RT-PCR
analyses of a quantitative RNA standard from BEI Resources (Cat. No. NR-52358). Error bars are
± one standard deviation. The linear regression results for N1 and N2 assays based on the average
results from duplicate 10-fold dilution series are shown.
4.4.2 RV-RT-PCR Experiments with SARS-CoV-2 Dilutions
A RV-RT-PCR experiment was conducted with SARS-CoV-2 and Vero E6 cells to determine an
optimal viral infection period and post-infection incubation period. Cells were added to 96-well plates
at 35,000 cells/well and incubated overnight in DMEM with 10% FBS at 37°C with 5% CO2 in a
humidified incubator. The SARS-CoV-2 stock previously titered at 1.88 x 106 plaque forming units
(PFU) per mL was serially diluted 10-fold in DMEM with 2% FBS. The following dilutions were added
to six replicate wells per dilution level per timepoint, using 0.1 mL per well: 10"2, 10"3, 10"4, and 10"5, as
38

-------
well as a negative control (medium only). SARS-CoV-2 was allowed to infect for either 2 hr or 4 hr at
37°C with 5% CO2. After this time, the medium was removed, and 0.1 mL fresh maintenance medium
(with 2% FBS) were added to each well and removed to wash away any virus that had not infected the
cells. Then, 0.1 mL fresh 2% FBS medium was added and the plates were incubated at 37°C with 5%
CO2 in a humidified incubator. At time 0 (To) and at each post-infection incubation period [12 hr (T12)
and 24 hr (T24)], the medium was removed and 0.1 mL PBS were added to each well. The plate was
then sealed and frozen at -80°C until RNA extraction was performed. RT-PCR was performed using the
CDC N1 assay as stated earlier. In parallel, the titer of the viral stock used to make dilutions was
determined via triplicate TCID50 analyses.
There were some issues with extraction and analysis of the 4-hr-infection sample wells; however, the
results for the 2-hr infection period for dilutions 10"3 to 10"5 (-220-2 PFU per sample well) are shown in
Table 26. Results for the negative control were non-detect (data not shown). The Ct data for To were
quite low (avg. Ct of -29 for -220 PFU/well and -28-34.5 for -22 PFU/well) showing significant
infection and possible viral propagation inside the host cells during the 2-hr infection period. With 12-
hr post-infection incubation, there was substantial viral propagation resulting in Ct values ranging from
-15.5 to 18 for the -220 PFU/well level and -18 to 19 for the -22 PFU/well level. Therefore, relatively
high ACt values of-12.5-13 were observed, even with low To Ct values. For the -2 PFU/well level,
T12 Ct values ranged from -19.5 to 24.6. Because the To Ct values for this PFU/well level were non-
detect, an average ACt value was -23 for 12-hr incubation. The T24 Ct values for this viral level were
lower than T12 Ct values (-5 Ct units) for 2 of 3 replicates, although one of the replicates showed higher
Ct values, which could be due to experimental error. Together these data for 2-hr infection and 12-hr
post-infection incubation indicated it was possible to shorten the infection period to 1-hr and still have
effective viral infection. Furthermore, the ACt results suggested the post-infection incubation period
could be shortened from 12 hr.
39

-------
Table 26 RV-RT-PCR Results for SARS-CoV-2-Infected Vero E6 Cells with 2-hr Infection
- First Experiment
Ksliiiiiilod
Pi t /
Siimplo
Replicitle
RT-I'CR
Rcplicitlc
C'i hv Post-1 n loci ion 1 nciihiit ion
l iinopoint After 2-hr Infection1
A\g. AC,
(Si))-¦¦¦¦¦¦
Well
I",,
1,2
1 24
1,2
1 24


1
28.4
17.7
12.2



1
2
28.7
17.5
12.0



3
28.6
17.9
12.2




Avg. (SD)
28.6 (0.2)
17.7 (0.2)
12.1 (0.1)




1
29.0
15.4
12.6



2
2
28.7
15.6
12.6
12.5
(0.1)
15.2
(0.1)
220
3
29.0
15.5
12.6


Avg. (SD)
28.9 (0.1)
15.5 (0.1)
12.6 (0.05)


1
29.1
15.8
16.2



3
2
29.0
15.9
16.3



3
28.9
15.8
16.2




Avg. (SD)
29.0 (0.1)
15.8 (0.04)
16.2 (0.1)



(hcniM
A\ g. (SI))
28.8 (0.2)
16.3 (1.0)
13.6(1.9)




1
31.9
18.5
13.2



1
2
32.4
18.5
13.3



3
32.7
18.6
13.4




Avg. (SD)
32.3 (0.4)
18.5 (0.1)
13.3 (0.1)




1
27.9
18.5
17.8



2
2
27.8
18.6
17.8
13.0
(4.1)
15.7
(4.4)
22
3
27.8
18.5
17.8


Avg. (SD)
27.9 (0.1)
18.5 (0.1)
17.8 (0.03)


1
34.6
18.9
16.6



3
2
34.8
18.8
16.7



3
34.1
18.8
16.7




Avg. (SD)
34.5 (0.4)
18.8 (0.1)
16.6 (0.1)



(hci-iill
A\ g. (SI))
31.6(2.9)
18.6 (0.2)
15.9(2.0)


* Values are based on dilution of the SARS-CoV-2 titered stock (TCID50 = 105 5 per 0.1 mL or 2.2 x 105 PFU/0.1 mL).
Estimated PFUAVell = TCID50/Well (corrected for dilution) x 0.7. TCID50/0.1 mL with SD = 2.19-4.56 x 105 and PFU/0.1
mL with SD = 1.54-3.19 x 105.
** Avg. and SD are from triplicate RT-PCR analyses per sample replicate. The Overall Avg. and SD are based on three
replicate samples.
*** SD represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the Tfvalues squared)/2, where Tf equals Ti2orT24.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. = Average; SD =
Standard Deviation.
40

-------
Table 26 (continued) RV-RT-PCR Results for SARS-CoV-2-Infected Vero E6 Cells with 2-hr
Infection - First Experiment
Ksliiiiiilod
PI-1 /Well1
Snmple
Replicnte
R1-PC K
Replic;ile
C'i hv Post-111 loclion 1 lieu bill ion
Timcpoinl Alter 2-hr Infection
A>«.
(SI))
AC,
In
Ti:
1 24
1,2
1 24


1
ND
19.4
13.7



1
2
ND
19.5
13.6



3
ND
19.6
13.5




Avg. (SD)
ND (NA)
19.5 (0.1)
13.6 (0.1)




1
ND
24.6
19.2



2
2
37.4
24.5
19.3
23.0
(1.6)
23.6
(5.7)
2
3
ND
24.5
19.3


Avg. (SD)
ND (NA)
24.6 (0.1)
19.3 (0.1)


1
41.8
21.8
33.6



3
2
ND
21.8
27.1



3
ND
21.9
33.3




A\«. (SI))
M) (NA)
2I.S (O.I)
31.3(3.7)



(hcrsill A\». (SI))
M) (NA)
22.0 (2.2)
21.4 (S.O)


* Values are based on dilution of the SARS-CoV-2 titered stock (TCID50 = 105 5 per 0.1 mL or 2.2 x 105 PFU/0.1 mL).
Estimated PFUAVell = TCID50/Well (corrected for dilution) x 0.7. TCID50/0.1 mL with SD = 2.19-4.56 x 105 and
PFU/0.1 mL with SD = 1.54-3.19 x 105.
** Avg. and SD are from triplicate RT-PCR analyses per sample replicate. ND is set to 45 to calculate an average Ct and
ACt. For 1 of 3 positive RT-PCR replicates, the sample result is also considered ND. The Overall Avg. and SD are based on
three replicate samples.
*** SD represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the Tfvalues squared)/2, where Tf equals Ti2orT24.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. = Average; SD =
Standard Deviation; ND = Non-Detect; NA = Not Applicable.
Another RV-RT-PCR experiment was conducted with SARS-CoV-2 and Vero E6 cells to determine the
optimum viral infection period and post-infection incubation period. In this case, 1-hr and 2-hr infection
periods were tested. The 96-well plates were seeded with cells as described above; however, cells were
infected with dilutions (10"3, 10"4 and 10"5) from a different SARS-CoV-2 stock, for which a
significantly lower average TCID50 value (10425 per 0.1 mL) was measured.
The results for the 1-hr and 2-hr infection periods for the 10"3 dilution (-12 PFU/well level) are shown in
Table 27 and Table 28, respectively. The average T12 ACt values for both infection periods were low
and did not meet the algorithm requirement of ACt > 6 (when factoring in SD), the ACt value was 7.0 ±
1.8 for 2-hr infection compared to 6.4 ±1.5 for 1-hr infection. For T24, the 2-hr infection showed an
average ACt of 12.1 ± 2.4, which did meet the algorithm requirement, while the 1-hr infection average
ACt of 7.4 ±1.7 did not meet the requirement with the standard deviation. These data suggested that 1-
hr infection was not sufficient to provide optimal detection using the RV-RT-PCR method, especially
for low starting viral levels.
41

-------
Table 27 RV-RT-PCR Results for SARS-CoV-2-Infected Vero E6 Cells with 1-hr Infection -
Second Experiment
Ksliimilcd
PI 1 /
S;i ill pic
Rcplicittc
R1-PC R
Rcplicitle
C'i hv Post-1 nleclion 1 ncnh;ition
liinopoi nt A Tier l-lir Infection
A\». AC 'i
(si) )••••••••
Well
In
Ti:
1 24
Ti:
1 24


1
33.2
25.1
23.5



1
2
33.4
25.0
23.4



3
33.2
25.1
22.6




Avg. (SD)
33.3 (0.1)
25.1 (0.1)
23.2 (0.5)




1
32.4
24.7
24.6



2
2
32.6
24.6
24.1
6.4
(1.5)
7.4
(1.7)
12
3
32.4
24.6
24.0


Avg. (SD)
32.5 (0.1)
24.7 (0.03)
24.2 (0.3)


1
31.9
28.7
29.2



3
2
32.0
28.8
26.3



3
32.0
28.9
28.7




Avg. (SD)
32.0 (0.1)
28.8 (0.1)
28.1 (1.5)



(>\er8lll
A\«. (SI))
32.6 (O.ft)
26.2 (2.0)
25.2 (2.4)


* Values are based on dilution of the SARS-CoV-2 titered stock (TCID50 = 104 25 per 0.1 mL or 1.24 x 104 PFU/0.1 mL),
from a different viral stock than the first replicate expt. Estimated PFUAVell = TCID50/Well (corrected for dilution) x 0.7.
TCID50/0.1 mL with SD = 1.22-2.60 x 104 and PFU/0.1 mL with SD = 0.85-1.82 x 104.
** Avg. and SD are from triplicate RT-PCR analyses per sample replicate. The Overall Avg. and SD are based on three
replicate samples.
*** SD represents the pooled SD, which equals the square root of the following: (SD for To values squared plus the SD for
the Tfvalues squared)/2, where Tf equals Ti2orT24.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. = Average; SD =
Standard Deviation.
42

-------
Table 28 RV-RT-PCR Results for SARS-CoV-2-Infected Vero E6 Cells with 2-hr Infection -
Second Experiment
Ksliimilcd
PI 1 /
Siimplo
Rcplicitlc
R1-PC R
Uopliciile
C'i hv Posl-111 Icclion 1 iicnh;ition
l iinopoint Al'lcr 2-hr Infection" •
A\g. AC 1
(SD)
Well
In
Ti:
1 24
1,2
1 24


1
33.4
23.5
20.0



1
2
32.7
23.2
20.3



3
32.6
23.6
20.1




Avg. (SD)
32.9 (0.4)
23.4 (0.2)
20.1 (0.2)




1
33.6
27.9
25.8



2
2
33.2
27.7
25.7
7.0
(1.8)
12.1
(2.4)
12
3
33.5
ND
25.9


Avg. (SD)
33.4 (0.2)
27.8 (0.1)a
25.8 (0.1)


1
34.0
28.4
18.3



3
2
34.8
29.5
18.3



3
34.0
27.0
18.9




Avg. (SD)
34.3 (0.5)
28.3 (1.2)
18.5 (0.3)



(hersill
.^«.(SI))
33.5 (0.7)
26.5(2.4)
21.5(3.3)


* Values are based on dilution of the SARS-CoV-2 titered stock (TCID50 = 104 25 per 0.1 mL or 1.24 x 104 PFU/0.1 mL).
Estimated PFUAVell = TCID50/Well (corrected for dilution) x 0.7.
** Avg. and SD are from triplicate RT-PCR analyses per sample replicate. The Overall Avg. and SD are based on three
replicate samples.
*** SD represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the Tfvalues squared)/2, where Tf equals Ti2orT24.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. = Average; SD =
Standard Deviation; ND = Non-Detect.
A third RV-RT-PCR experiment was conducted with SARS-CoV-2 suspension and Vero E6 cells to
further determine the viral infection period (1-hr vs. 2-hr) and post-infection incubation period, and to
evaluate the initial method sensitivity of detection. In this case, a new SARS-CoV-2 stock was used.
This stock had a titer of 105 25 TCID50 per 0.1 mL and several vials were prepared and stored at -80°C.
During quantitation of this stock as well as the other two stocks used for previous experiments, it was
noted that the TCID50 results could change slightly after 4 days (of cell-culture-virus incubation),
therefore final values for viral titers were determined after 6-8 days.
For the experiment, 96-well plates were seeded with cells as described and were then infected with
SARS-CoV-2 dilutions (10"3, 10"4 and 10"5) or had medium added for negative control wells. The
infection period was either 1-hr or 2-hr, after which plates were processed as described (Section 2.5).
The results for the 2-hr infection period are shown in Table 29. Results for the negative control wells
were non-detect (data not shown).
The To Ct values were comparable to those for similar starting viral levels, -220 PFU/well for the first
experiment vs. -124 PFU/well for this experiment, with average values of-28.6-29 and -28.4-29.3,
respectively. For a 10-fold lower viral levels, To Ct values were -28-34.5 for the first experiment (-22
PFU/well) and -32 for this experiment (-12 PFU/well). For the 124 PFU/well level, 3 of 3 sample
replicates showed low T12 Ct values and an average ACt of 13.7 ± 0.6. For 24-hr incubation, the
43

-------
average ACt value was 18.7 ± 0.6. These average ACt values compared to 12.5 ±0.1 and 15.2 ± 0.2 for
12- and 24-hr incubation, respectively for the -220 PFU/well level in the first experiment. For the 10"4
dilution (-12 PFU/well) for this experiment, 2 of 3 average T12 Ct values were low, -18.5-21 with the
third sample at an average Ct value of 34.8. Therefore the average ACt value 12.3 ± 0.8 was calculated
based on two T12 Ct values. At T24, all 3 sample replicates had Ct values ranging from -12.7-15.9 and
the average ACt value was 17.4 ± 0.8. Together the results showed some variability at low viral levels
with 3 of 3 positive replicates for -2 PFU/well in one experiment and 2 of 3 positive for -12 PFU/well
in the replicate experiment.
The RV-RT-PCR results for the 1-hr infection period are shown in Table 30. Negative control wells
were non-detect (data not shown). The average ACt values for the 10"4 dilution (-124 PFU/well) were
11.5 + 0.3 for 12-hr incubation and 16.8 ± 0.8 for 24-hr incubation. For both incubation time periods, 3
of 3 replicates had low Ct values of-17-18 for 12-hr incubation and -11.5-14 for 24-hr incubation. For
the 10"4 dilution (-12 PFU/well), only 2 of 3 sample replicates showed lower Ct values for both 12-hr
and 24-hr incubation, therefore the average ACt value was calculated based on two T12 or T24 values as
12.7 ±2.1 and 21.3 ± 2.0, respectively.
Based on the data for 1-hr and 2-hr infection periods, a 2-hr infection period was selected for further
experiments on the SARS-CoV-2 RV-RT-PCR method development. The 2-hr infection period showed
slightly lower Ct values for sample replicates than for 1-hr infection and represented a more
conservative method for actual environmental samples, which may contain low viral levels as well as
interferents that negatively impact viral infection. A summary of results for the first and third replicate
experiments for the 2-hr infection period are shown in Table 31.
44

-------
Table 29 RV-RT-PCR Results for SARS-CoV-2-Infected Vero E6 Cells with 2-hr Infection -
Third Experiment
Kstiniiiled
Pi t /
Siimplo
Ueplic;ite
RT-I'CR
kepliciite
C'i hv I'osl-lnfeclion Incubillion
l imopoint After 2-hr Infection1
A\». AC 'i
(Si)) ••••••••••
Well*
I",,
Ti:
1 24
Ti:
1 24


1
29.1
14.4
9.5



1
2
29.4
14.4
9.6



3
29.2
14.4
9.6




Avg. (SD)
29.2 (0.1)
14.4 (0.01)
9.6 (0.1)




1
28.3
16.2
10.1



2
2
28.5
16.2
10.2
13.7
(0.6)
18.7
(0.6)
124
3
28.4
16.2
10.1


Avg. (SD)
28.4 (0.1)
16.2 (0.03)
10.1 (0.03)


1
29.4
15.4
11.0



3
2
29.1
15.3
11.0



3
29.4
15.4
11.2




Avg. (SD)
29.3 (0.2)
15.3 (0.04)
11.1 (0.1)



OmtsiII A\». (SI))
29.0 (0.4)
15.3 (0.8)
10.3(0.7)




1
32.6
18.4
12.8



1
2
32.1
18.5
12.6



3
31.6
18.5
12.8




Avg. (SD)
32.1 (0.5)
18.5 (0.1)
12.7 (0.1)




1
32.6
21.2
15.6



2
2
31.9
21.2
15.6
12.3
(0.8)a
17.4
(0.8)
12
3
31.7
21.2
15.6


Avg. (SD)
32.1 (0.5)
21.2 (0.03)
15.6 (0.01)


1
32.4
34.4
16.0



3
2
32.2
34.8
15.9



3
32.1
35.2
15.8




A\«. (SD)
32.3(0.1)
34.8 (0.4)
15.9(0.1)



OmtsiII A\». (SI))
32.1 (0.3)
19.8 (1.4) '
14.7(1.5)


* Values are based on dilution of the SARS-CoV-2 titered stock (TCID50 = 105 25 per 0.1 mL or 1.24 x 105
PFU/0.1 mL) from a different viral stock than the first or second replicate expt. Estimated PFUAVell =
TCID50/Well (corrected for dilution) x 0.7. TCID50/0.1 mL with SD = 1.22-2.60 x 105 and PFU/0.1 mL with SD
= 0.85-1.82 x 105.
** Avg. and SD are from triplicate RT-PCR analyses per sample replicate. The Overall Avg. and SD are based on
three replicate samples except where noted.
*** SD represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the
SD for the Tf values squared)/2, where Tf equals Ti2or T24.
a Overall Avg. CT and SD, and Avg. ACT and SD based on 2 positive replicate samples; one replicate sample had
higher Ct values (-34-35) and was not included in the average and SD or pooled SD.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; CT = Cycle Threshold; Avg. = Average; SD =
Standard Deviation.
45

-------
Table 30 RV-RT-PCR Results for SARS-CoV-2-Infected Vero E6 Cells with 1-hr Infection -
Third Experiment
Ksliimitcil
PI 1 /Well
Siimplo
Rep li title
RT-I'CR
Rcpliciilc
C'i hv I'osl-lnlcclion Incubillion
Tinicpoinl Alter l-lir In feci ion
A\«. AC
(Si))
I",,
1,2
1 24
Ti:
1 24


1
29.2
17.6
11.4



1
2
29.2
17.5
11.5



3
29.0
17.4
11.4




Avg. (SD)
29.1 (0.1)
17.5 (0.1)
11.4 (0.1)




1
29.5
18.3
13.9



2
2
29.6
18.3
14.0
11.5
(0.3)
16.8
(0.8)
124
3
29.4
18.3
13.8


Avg. (SD)
29.5 (0.1)
18.3 (0.02)
13.9 (0.1)


1
29.3
17.8
12.5



3
2
29.5
17.8
12.4



3
29.6
17.8
12.4




A\«. (SI))
29.5 (0.2)
17.8(0.01)
12.4(0.04)



(heiiill A\». (SI))
29.4(0.2)
17.9 (0.4)
12.6 (I.I)




1
33.5
21.8
14.7



1
2
33.8
22.2
14.6



3
34.1
22.3
14.6




Avg. (SD)
33.8 (0.3)
22.1 (0.3)
14.7 (0.1)




1
37.4
38.9
14.9



2
2
38.1
37.7
14.9
12.7
(2.1)a
21.3
(2.0)a
12
3
35.6
37.1
14.6


Avg. (SD)
37.1 (1.3)
37.9 (0.9)
14.8 (0.2)


1
36.2
24.4
36.3



3
2
39.0
24.7
34.6



3
36.1
24.2
34.4




Avg. (SD)
37.1 (1.7)
24.4 (0.3)
35.1 (1.1)



(heiiill A\». (SI))
36.0(2.0)
23.3 (1.0) '
14.7 (0.1);'


* Values are based on dilution of a SARS-CoV-2 titered stock (TCID50 = 105 25 per 0.1 mL or 1.24 x 105 PFU/0.1 mL), from
a different viral stock than the first or second replicate expt. Estimated PFU/Well = TCID50/Well (corrected for dilution) x
0.7. TCID50/0.1 mL with SD = 1.22-2.60 x 105 and PFU/0.1 mL with SD = 0.85-1.82 x 105.
** Avg. and SD are from triplicate RT-PCR analyses per sample replicate. The Overall Avg. and SD are based on
three replicate samples except where noted.
*** SD represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the Tfvalues squared)/2, where Tf equals Ti2orT24.
a Overall Avg. CT and SD, and Avg. ACT and SD based on 2 positive replicate samples; one sample replicate had high CT
values (-34-36 or -37-39) and was not included in the average and SD or pooled SD.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. = Average; SD =
Standard Deviation.
46

-------
Table 31 Summary of RV-RT-PCR Avg. ACt Results from Replicate Experiments*
for SARS-CoV-2-Infected Vero E6 Cells with 2-hr Infection
Estimated
PFU/Well
T12 Avg. ACt
(SD)
T12 Positive Results
T24 Avg. ACt
(SD)
T24 Positive Results
220
12.5 ±0.1
3 of 3
15.2 ±0.2
3 of 3
124
13.7 ± 0.6
3 of 3
18.7 ± 0.6
3 of 3
22
13.0 ±4.1
3 of 3
15.7 ± 4.4
3 of 3
12
12.3 ±0.8
2 of 3
17.4 ±0.8
3 of 3
2
23.0 ± 1.6
3 of 3
23.0 ±5.7
3 of 3
* The results for the first and third experiments are shown; the second experiment was omitted from the summary
due to variability in cell culture conditions and the viral stock used.
PFU = Plaque Forming Units; Ct = Cycle Threshold; Avg. = Average; SD = Standard Deviation.
From the experiments with SARS-CoV-2 suspension, the RV-RT-PCR method showed detection (ACt
> 6) for 3 of 3 sample replicates at -22 PFU/well with a 2-hr infection period and 12-hr post-infection
incubation period. Some variability in results was observed between experiments at lower viral levels
using the same infection and incubation periods (2-hr and 12-hr, respectively). In one experiment, the
-12 PFU/well level showed 2 of 3 positive sample replicates, whereas, in another experiment the -2
PFU/well level gave 3 of 3 positive sample replicates. In all cases, T12 ACt values for positive samples
were quite high, ranging from 12.3 to 23. As discussed, because these experiments were done with viral
suspension prepared as 10-fold dilutions, with 0.1 mL added to replicate sample wells with different
plates for each timepoint (To, T12 and T24), the results are reported for estimated PFU/well. Therefore, to
express the results for PFU/sample, the estimated PFU/well (i.e., PFU/0.1 mL) would be doubled and 22
PFU/well would be 44 PFU/sample. Thus, the data showing <50-virion sensitivity of detection was
achieved for these test conditions. The conditions established in the viral suspension experiments (cell
seeding density, infection period, etc.) were used in subsequent swab swatch experiments to establish an
optimum post-infection incubation period.
4.4.3 RV-RT-PCR analysis of SARS-CoV-2-spiked swab swatches
As described for experiments with MHV, initial RV-RT-PCR experiments with SARS-CoV-2 were also
conducted using swab swatches since Puritan™ swabs suggested by the CDC to sample surfaces for
viral contamination continued to be unavailable. As discussed, the swatches used in this study were
made with the same swab material, which was supplied by Puritan.
Using the 2-hr infection incubation period (and Vero E6 seeding cell density of 35,000 cells) as
established with the SARS-CoV-2 suspension, RV-RT-PCR experiments were performed with spiked
swab swatches. Briefly, the swatches (-5 cm2) were placed in sterile 50-mL conical tubes, pre-wet with
1.5 mL PBS, and spiked with 0.5 mL SARS-CoV-2 stock, diluted 10-fold to starting viral titers
including 10"2 (-2 logio), 10"3 (-3 logio), and a 2,000-fold (-3.3 logio) dilution, as well as a negative
control (no virus), with triplicates per viral level. Viral recovery efficiency was also evaluated using
TCID50 analysis with replicate swatches spiked with either the 10"2 or 10"3 dilution in triplicate. Swab
swatches were processed as described previously (Section 2.6). The UF retentate (-0.3 to 0.5 mL) was
adjusted to 0.5 mL with PBS, and filtered through an 0.22-micron spin filter. The filtrate for each
swatch was split into 0.1 mL aliquots and added to a separate cell culture plate for each timepoint, To,
T9, T12, and T24, followed by addition of 0.1 mL cell culture medium (with 2% FBS) to each well. After
a 2-hr infection period, the viral inoculum was removed, fresh medium was added and removed to wash
47

-------
any residual vims, and fresh growth medium (with 2% FBS) was added to each well. At each timepoint,
the medium was removed, PBS was added and the plate was sealed with aluminum adhesive seal, and
stored at -80°C until the sample wells were extracted for RNA and analyzed by RT-PCR using the
SARS-CoV-2 N1 assay as previously described.
The results for viral recovery efficiency testing are shown in Table 32. The recovery efficiency value of
100% is not included in the calculation of average percent recovery (one replicate for 620 PFU/swatch
level), because this value is inconsistent with the other swatch replicates, and is likely due to error in
processing and analysis. The average recoveries were 22.4% for the -8,900 PFU/swatch level and
~43.9% for the -890 PFU/swatch level. The average of these two values, 33.2% was used to estimate
the starting PFU/well for the -310 PFU/swatch level.
Table 32 SARS-CoV-2 Recovery from Swab Swatch Samples
Ksliniiilcil
Stiirlin<> Pl-'l /
Swsilch*
Swsilch
Ucplicsilc
Rcco\ civil
Pl-'l /
Swsilch" -
I'crccnl
Ucc<>\ crv
(%)
Kslimsilctl
Pl-'l / W ell1
6200
1
1968
31.6
-280
2
1107
17.8
3
1107
17.8
A\«. (SI))
1394 (497)
22.4 (8.0)
620
1
620
100
-55
2
197
31.6
3
350
56.2
A\«. (SI))
390 (216)
43.9 (17.4)

A\». Pcrccnl Kccomtv
33.2

310

Not Determined
Not Determined
-21
* Values are based on dilution of the SARS-CoV-2 titered stock (TCID50 = 105 25 per 0.1 mL or 1.24 x 105 plaque forming
units (PFU)/0.1 mL) with 0.5 mL used to spike the swatch. TCID50/0.1 mL with SD = 1.12-2.81 x 105 and PFU/0.1 mL with
SD = 0.79-1.97 x 105.
** Values are based on TCID50 analysis of swatch UF-retentates from sample processing. TCID50 values showing >100%
recovery were not included in calculation of the average recovery (see text for explanation).
*** Values are based on 0.1 mL used per well for each plate (T0, T9, Tu, and T24) from the total 0.5 mL from each spiked
swatch. The average percent recovery was used to calculate the estimated PFU/well for swatches spiked with -445 PFU. Not
Determined; Recovery efficiency was not determined directly from these viral dilutions.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Avg. = Average; SD = Standard Deviation.
RV-RT-PCR results from the first experiment are shown in Table 33 with the average TCID50 data
from replicate swatches in the first column. For the 10"2 dilution (-280 FPU/well), 3 of 3 swatch
replicates were detected for both 9- (T9) and 12-hr (T12) post-infection incubation periods, with average
ACt values of 10.3 and 15.0 for T9 and T12, respectively. Likewise for the 10"3 dilution (-55 PFU/well)
and -3.3 logio dilution (-21 PFU/well), there were 3 of 3 positive swatch replicates with average ACt
values for T9 ranging from 9.5 to 12.2, significantly above the criterion of ACt > 6 for positive
detection. In addition, for these two lower PFU/well levels, there were 3 of 3 positive swatch replicates
with average ACt values of-14.4 to 15.8. Results for the negative control swatches were non-detect
(data not shown). In Table 34, the data are summarized as average Ct and average ACt values, which
included triplicate RT-PCR analyses for each triplicate swatch.
48

-------
Table 33 RV-RT-PCR Results for SARS-CoV-2-Spiked Swab Swatches Processed and Used to
Infect Vero E6 Cells with 2-hr Infection
Ksliiiiiilod
PI I /
Swsilch
Replicate
R'I'-IH'R
Replicate
Ci hv Post-In loci ion 1 n en h;it ion
Timcpoint Al'lcr 2-hr Infection1
A\». AC
(SI))'""""
Well
In
1,
1,2
1,
T,:


1
28.7
18.5
13.8



1
2
28.6
18.6
14.1
10.1
14.6

3
28.6
18.6
14.0
(0.04)
(0.1)


Avg. (SD)
28.6 (0.05)
18.6 (0.03)
14.0 (0.2)




1
27.9
17.8
12.9




2
28.0
17.6
12.9
10.2
15.0
280
2
3
27.9
17.8
13.0
(0.1)
(0.04)


Avg. (SD)
27.9 (0.04)
17.7 (0.2)
12.9 (0.1)




1
29.9
19.1
14.5




2
29.8
19.2
14.5
10.8
15.3

3
3
30.0
19.0
14.6
(0.1)
(0.1)


Avg. (SD)
29.9 (0.1)
19.1 (0.1)
14.5 (0.02)



(herall .
(SI))
28.8 (0.9)
18.5 (0.6)
13.8 (0.7)
10.4 (0.4)
15.0 (1.0)


1
31.0
19.4
io:



1
2
31.5
19.3
16.3
12.2
15.2

3
31.4
19.3
16.4
(0.08)
(0.1)


Avg. (SD)
31.5 (0.1)
19.3 (0.03)
16.3 (0.1)




1
31.5
20.4
17.0



2
2
31.5
20.5
17.1
11.1
14.4
55
3
31.5
20.4
17.1
(0.06)
(0.05)


Avg. (SD)
31.5 (0.05)
20.4 (0.1)
17.1 (0.1)




1
32.3
22.7
16.9



3
2
32.3
22.8
17.0
9.5
15.2

3
32.2
22.6
17.1
(0.08)
(0.1)


Avg. (SD)
32.2 (0.1)
22.7 (0.1)
17.0 (0.1)

therall.
^g. (SD)
31.8(0.4)
20.8(1.5)
16.8 (0.4)
11.0(1.1)
15.0 (0.5)
* Estimated PFUAVell is based on T(ll )N> ;in;il\ sis and \ nal iva>\ ci> of repliculc swatches (lahlc ^2). w here I'll I/Wcll
= TCID50AVell (corrected for dilution) x 0.7.
** Avg. and SD are from triplicate RT-PCR analyses per swatch replicate. The Overall Avg. and SD are based on
three replicate swatches.
*** SD represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the Tfvalues squared)/2, where Tf equals T9orTi2.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. = Average; SD =
Standard Deviation.
49

-------
Table 33 RV-RT-PCR Results for SARS-CoV-2-Spiked Swab Swatches Processed and Used to
Infect Vero E6 Cells with 2-hr Infection (continued)
Ksliiiiiilod
PI I /
Swjitch
Replicate
R'I'-IH'R
Replicate
C'i by Post-In loci ion Incubation
Timepoint Alter 2-hr Infection1
A\». AC
(SI))'-"""
Well
In
1,
1,2
1,
T,:


1
33.1
22.3
18.2



1
2
32.9
22.2
18.5
10.7
14.6

3
33.0
22.3
18.5
(0.06)
(0.1)


Avg. (SD)
33.0 (0.1)
22.3 (0.1)
18.4 (0.2)




1
32.8
21.0
16.7




2
32.9
21.1
16.7
11.5
15.8
21
2
3
31.9
20.9
16.8
(0.4)
(0.4)


Avg. (SD)
32.5 (0.6)
21.0 (0.1)
16.7 (0.05)




1
33.0
23.3
17.8




2
33.2
23.3
17.9
9.8
15.2

3
3
33.1
23.2
17.9
(0.09)
(0.1)


Avg. (SD)
33.1 (0.1)
23.3 (0.1)
17.9 (0.1)



(herall .
\\g. (SI))
32.9 (0.4)
22.2 (1.«))
17.7 (0.7)
10.7 (0.8)
15.2 (0.6)
* Estimated PFUAVell is based onTCID50 aiiaK sis and \ nal roan ci> of icpl icalc swale lies ilahlc "O i. w here HI J/Wcll
= TCID50/Well (corrected for dilution) x 0.7.
** Avg. and SD are from triplicate RT-PCR analyses per swatch replicate. The Overall Avg. and SD are based on
three replicate swatches.
*** SD represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the Tfvalues squared)/2, where Tf equals T9orTi2.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. = Average; SD =
Standard Deviation.
50

-------
Table 34 Summary of RV-RT-PCR Results for SARS-CoV-2-Spiked Swab Swatches Processed
and Used to Infect Vero E6 Cells with 2-hr Infection
Ksl i muled
PI 1 /Well-
S\\:ileh
Replicate
A>«>. (SI)
111c
A
)( Vhv Post-1111'eclion
11 h;ition Timepoint
ler 2-hr Inl'eelion
A\». AC'i (SI))1'-"""
111
T.,
Ti:
1,
Ti:
280
1
28.6(0.05)
18.6(0.03)
14.0 (0.2)
10.1 (0.04)
14.6 (0.1)
2
27.9 (0.04)
17.7 (0.2)
12.9(0.1)
10.2 (0.11)
15.0(0.04)
3
29.9 (0.1)
19.1 (0.1)
14.5 (0.02)
10.8 (0.1)
15.3 (0.1)
(henill
A\«. (SI))
2S.S («.»>)
18.5 (0.6)
13.8(0.7)
10.3 (1.0)
15.0 (1.0)
55
1
31.5 (0.1)
19.3 (0.03)
16.3 (0.1)
12.2 (0.08)
15.2 (0.1)
2
31.5 (0.05)
20.4 (0.1)
17.1 (0.1)
11.1 (0.06)
14.4 (0.05)
3
32.2 (0.1)
22.7 (0.1)
17.0(0.1)
9.5 (0.08)
15.2 (0.1)
(henill
A\«. (SI))
31.8 (0.4)
20.8 (1.5)
16.8(0.4)
11.0(1.1)
15.0 (0.5)
21
1
33.0 (0.1)
22.3 (0.1)
18.4 (0.2)
10.7 (0.06)
14.6 (0.1)
2
32.5 (0.6)
21.0 (0.1)
16.7 (0.05)
11.5 (0.4)
15.8 (0.4)
3
33 1 (ii |)
23 3 (() 1)
17 w (ii I)
w X (ii tw)
15 2 (() 1)
(henill
(SI))
32/) (0.4)
22.2 (1.0)
17.7(0.7)
10.7(0.8)
15.2 (0.6)
* Estimated I'll Well is basal on T(ll )N> ;m;il\ sis and \ nal ieeo\ ei> of icpl icalc swale lies (lahle '2). \\ here HI I/Wcll
= TCID50/Well (corrected for dilution) x 0.7.
** Data are from triplicate RT-PCR analyses of triplicate swatches. The Overall Avg. and SD are based on three replicate
swatches.
*** SD represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the Tfvalues squared)/2, where Tf equals T9orTi2.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. = Average; SD =
Standard Deviation.
A replicate RV-RT-PCR experiment was conducted using swab swatches. Briefly, pre-wet swatches had
0.5 mL of SARS-CoV-2 diluted to -2, -3, or -3.3 logio relative to the stock (1.6 x 105 PFU/0.1 mL)
added, triplicate per dilution, as well as triplicate negative control swatches with PBS added. As for the
first swab swatch experiment, to evaluate viral recovery efficiency, swatches were spiked with the 10"2
or 10"3 dilution in triplicate and analyzed by TCID50. Subsequent steps were as described previously.
After ultrafiltration and filtration resulting in -0.5 mL, aliquots of 0.1 mL were added to each timepoint
cell culture plate (To, T9, T12, and T24), with 0.1 mL medium then added to each well. Following a 2-hr
infection period and the appropriate post-infection incubation period (T9, T12, and T24), the plates were
processed, stored at -80°C, and then RNA was extracted from wells and analyzed by RT-PCR.
The results for viral recovery efficiency testing of replicate swatches are shown in Table 35. As
mentioned, the recovery efficiency value > 100% is not included in the calculation of average percent
recovery (one replicate for 436 PFU/swatch level), since it is inconsistent with the other swatch
replicates, and likely due to error in processing and analysis. The average recoveries were 35.2% for the
51

-------
-4,360 PFU/swatch level and -13.9% for the -436 PFU/swatch level. The average of these two values,
24.6% was used to estimate the starting PFU/well for the -218 PFU/swatch level. Because of recovery
efficiency variation, the estimated PFU/well for the two lower PFU/swatch levels were similar.
Table 35 SARS-CoV-2 Recovery from Swab Swatch Samples - Replicate Experiment
KsliniiiU'd
Stiirlin<> Pl-'l /
Swiilclr-
Swsilch
Rcpliciilc
Reco\ crcil
PI I /
Swsilch" "
I'crccnl
Rec<>\cry
<%)
Ksli milled
Pl-'l /
Well • •
6200
1
3500
56.2
-438
2
1970
31.6
3
1110
17.8
A\«.(SI»
2190(1210)
35.2 (l»>.5)
620
1
111
17.8
-17
2
>620
>100
3
62 2
10.0
A\«. (SI))
86(34)
13/) (5.5)

A\». I'crccnt Reco\erv
24.6

310

Not
Determined
Not
Determined
-15
* Values are based on dilution of the SARS-CoV-2 titered stock (TCID50 = 105 25 / 0.1 mL or 1.24
x 105 PFU/0.1 mL) with 0.5 mL used to spike the swatch. TCID50/0.1 mL with SD = 1.32-2.40 x
105 and PFU/0.1 mL with SD = 0.92-1.68 x 105.
** Values are based on TCID50 analysis of swatch UF-retentates from sample processing. TCID50
values showing >100% recovery were not included in calculation of the average recovery (see text
for explanation). Not Determined; Recovery efficiency was not determined directly from this viral
dilution.
*** Values are based on 0.1 mL used per well for each plate (T0, T9, Tu, and T24) from the total 0.5
mL from each spiked swatch. The average percent recovery was used to calculate the estimated
PFU/well for swatches spiked with -218 PFU.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Avg. = Average;
SD = Standard Deviation.
The RV-RT-PCR results for all three viral dilutions are shown in Table 36. The data for negative
controls were non-detect (data not shown). Since positive results were obtained for T9 and T12, the T24
plate (with all three dilutions) was not processed and analyzed. For the 10"2 dilution (-438 PFU/well), 3
of 3 swatch replicates were detected for T9 and T12 incubation periods with average ACt values from 6.6
to 10.3 and 12.6 to 14.6, respectively. However, for the -3 logio (-17 PFU/well) and -3.3 logio (-15
PFU/well) dilutions, 2 of 3 swatches had positive results for T9 incubation with ACt values ranging from
7.1 to 10.6, with other swatches showing ACt values of 4.5 and 3.9 for -17 PFU/well and -15 PFU/well,
respectively, which did not meet the requirement for ACt> 6. For T12 incubation period, the 10"3
dilution (-17 PFU/well) had 3 of 3 swatches with positive results with average ACt of 13.0, whereas the
—3.3 logio dilution (-15 PFU/well) showed 2 of 3 positive with ACt values of 11.1 and 13.5. In Table
37, the data are summarized as average Ct and average ACt values that included triplicate RT-PCR
analyses for each swatch replicate.
Variability between replicate swatches for low viral levels and poor recovery efficiencies (Table 35)
may have contributed to 2 of 3 replicate swatches showing positive RV-RT-PCR results. In addition, it
52

-------
was noted that the Vero E6 cell culture wells were less confluent than the previous experiment, which
could have negatively affected viral infection and propagation. When compared to the high T12 ACt
values (11.1-13.5) for other swatch replicates for the same viral level, this replicate could have been the
result of technical error (i.e., pipetting variability).
Table 36 RV-RT-PCR Results for SARS-CoV-2-Spiked Swab Swatches Processed and Used to
Infect Vero E6 Cells with 2-hr Infection - Replicate Experiment
KsliiiiiiU'd
PI 1 /
Swsilch
Rcplic;ilc
R'I'-IH'R
Rcpliciilc
Ci hy Posl-111 loclion Incubiilion
l iniopoint Alter 2-hr Infection'-"
A\». AC
(SI)) •• ••
Well
1 „
1,
1,2
1,
T,:


1
27.4
17.0
12.7



1
2
27.0
16.9
12.6
10.3
14.6

3
ND
16.9
12.6
(0.2)
(0.2)


Avg. (SD)
27.2 (0.3)a
16.9 (0.05)
12.6 (0.1)




1
26.7
18.3
14.4



2
2
26.8
ND
14.2
8.4
12.6
438
3
27.0
18.5
13.9
(0.2)
(0.2)


Avg. (SD)
26.8 (0.1)
18.4 (0.2)a
14.2 (0.2)




1
26.8
20.4
14.5



3
2
26.9
20.4
14.3
6.6
12.7

3
27.3
20.4
14.3
(0.1)
(0.2)


Avg. (SD)
27.0 (0.2)
20.4 (0.03)
14.3 (0.1)



(hoiiill .
(SI))
27.0 (0.3)
18.6 (1.5)
13.7 (0.8)
8.4(1.1)
13.3(0.6)


1
30.0
2 0.4
16.9



1
2
30.2
20.3
17.4
9.8
13.1

3
ND
20.2
16.6
(0.1)
(0.3)


Avg. (SD)
30.1 (0.1)a
20.3 (0.1)
17.0 (0.4)




1
29.6
22.7
17.9



2
2
30.1
22.7
ND
7.1
12.3

3
30.3
23.2
17.6
(0.4)
(0.3)
17

Avg. (SD)
30.0 (0.4)
22.9 (0.3)
17.7 (0.2)a




1
30.0
25.7
16.9



3
2
31.1
26.3
17.1
4.5
13.7

3
30.6
26.2
16.6
(0.4)
(0.4)


Avg. (SD)
30.6 (0.5)
26.1 (0.3)
16.9 (0.3)



(hoiiill A\». (SI))
(3 Reps)
30.2 (0.4)
23.1 (2.5)
17.2 (0.5)
7.1 (1.8)
13.0 (0.4)

(herall A\». (SI))
(2 Reps)1'
30.1 (0.3)
21.6 (I.I)
17.4(0.5)
8.5(1.1)
12.7(0.4)
* Estimated PFUAVell is based on TCID50 analysis and viral % recovery of replicate swatches (Table 35), where PFUAVell
= TCID50/Well (corrected for dilution) x 0.7.
** Avg. and SD are from triplicate RT-PCR analyses per swatch replicate. The Overall Avg. and SD data are
based on replicate swatches.
53

-------
*** SD represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the Tfvalues squared)/2, where Tf equals T9orTi2.
a Avg. Ct and SD are based on 2 positive RT-PCR analyses; one replicate was non-detect.
b Avg. ACt and SD are based on replicate swatches 1 and 2; the 3rd swatch replicate had higher CT values (~26) at T9 and was
not included in the average and SD or pooled SD.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. = Average; SD =
Standard Deviation; ND = Non-Detect; Reps = Replicates.
Table 36 RV-RT-PCR Results for SARS-CoV-2-Spiked Swab Swatches Processed and Used to
Infect Vero E6 Cells with 2-hr Infection - Replicate Experiment (Cont'd)
Ksl i muled
PI-1 /
Swiilch
Repliciile
RT-PCR
Repliciile
C'i hy Post-Infection Inciihiition
l imepoint Alter 2-hr Infection1-
A\j». AC,
(SI) )••••• ••
Well
I",,
1,
1,2
1,
T,:


1
31.3
27.4
32.9



1
2
31.1
27.2
32.5
3.9
-1.4

3
30.8
27.1
32.7
(0.2)
(0.2)


Avg. (SD)
31.1 (0.2)
27.2 (0.2)
32.7 (0.2)




1
30.6
20.2
19.4



2
2
30.9
20.3
19.9
10.6
11.1

3
30.8
19.9
19.8
(0.2)
(0.3)
15

Avg. (SD)
30.8 (0.2)
20.2 (0.2)
19.7 (0.3)




1
ND
21.4
16.7



3
2
30.4
21.1
17.0
9.3
13.5

3
30.4
21.0
17.0
(0.1)
(0.1)


Avg. (SD)
30.4 (0.03)a
21.1 (0.2)
16.9 (0.1)



OmtsiII A\». (SI))
(3 Reps)
30.7 (0.3)
22.S (3.3)
23.1 (7.3)
7.9(2.4)
7.6 (5.2)

(heriill A Mi. (SI))
(2 Reps)1'
30.6 (0.2)
20.7 (0.5)
18.3 (1.2)
9.9 (0.4)
12.3(1.1)
* Estimated PFUAVell is based on TCID50 analysis and viral % recovery of replicate swatches (Table 35), where PFUAVell
= TCID50/Well (corrected for dilution) x 0.7.
** Avg. and SD are from triplicate RT-PCR analyses per swatch replicate. The Overall Avg. and SD data are based
on replicate swatches.
*** SD represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the Tfvalues squared)/2, where Tf equals T9orTi2.
a Avg. CT and SD based on 2 positive RT-PCR analyses; one replicate was non-detect.
b Avg. ACt and SD based on replicate swatches 2 and 3; the 1st swatch replicate had higher Ct values, ~27 and ~33 at T9 and
T12, respectively, and was not included in the average and SD or pooled SD.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. = Average; SD =
Standard Deviation; ND = Non-Detect; Reps = Replicates.
54

-------
Table 37 Summary of RV-RT-PCR Results for SARS-CoV-2-Spiked Swab Swatches Processed
and Used to Infect Vero E6 Cells with 2-hr Infection
list i 111 sited
IT I /
Well*
Swsilch
Replic.ile
A\». (SI)) C'l1" by Post-1 nI'eclion
Inciihiilion Timepoinl
Alter 2-hr Infection
A\». AC 'i (SI))
In
1,
T1;
1,
1
438
1
27.2 (0.3)
16.9 (0.05)
12.6 (0.1)
10.3 (0.2)
14.6 (0.2)
2
26.8 (0.1)
18.4 (0.2)
14.2 (0.2)
8.4 (0.2)
12.6 (0.2)
3
27.0 (0.2)
20.4 (0.03)
14.3 (0.1)
6.6(0.1)
12.7 (0.2)
\\». (SI))
27.0 (0.3)
18.6 (1.5)
13.7 (0.8)
8.4(1.1)
13.3 (0.6)
17
1
30.1 (0.1)
20.3 (0.1)
17.0 (0.4)
9.8 (0.1)
13.1 (0.3)
2
30.0 (0.4)
22.9 (0.3)
17.7 (0.2)
7.1 (0.4)
12.3 (0.3)
3
30.6 (0.5)
26.1 (0.3)
16.9 (0.3)
4.5 (0.4)
13.7 (0.4)
A\«>. (SI))
(3 Reps)
30.2 (0.4)
23.1 (2.5)
17.2 (0.5)
7.1 (1.8)
13.0(0.4)
A>«.(SD)
(2 Reps);i
30.1 (0.3)
21.6(1.1)
17.4 (0.5)
8.5(1.1)
12.7 (0.4)
15
1
31.1 (0.2)
27.2 (0.2)
32.7 (0.2)
3.9(0.2)
-1.4 (0.2)
2
30.8 (0.2)
20.2 (0.2)
19.7 (0.3)
10.6 (0.2)
11.1 (0.3)
3
30.4 (0.03)
21.1 (0.2)
16.9 (0.1)
9.3 (0.1)
13.5 (0.1)
A\«. (SI))
(3 Reps)
30.7 (0.3)
22.8 (3.3)
23.1 (7.3)
7.9(2.4)
7.6(5.2)
A\«>. (SI))
(2 Reps)1'
30.6 (0.2)
20.7 (0.5)
18.3 (1.2)
»>.»> (0.4)
12.3 (I.I)
* Estimated PFUAVell is based on TCID50 analysis and viral % recovery of replicate swatches (Table 35), where PFU/Well
= TCID50/Well (corrected for dilution) x 0.7.
** Data are from triplicate RT-PCR analyses of triplicate swatches.
*** SD represents the pooled SD, which equals the square root of the following: (SD for To values squared plus the SD for
the Tfvalues squared)/2, where Tf equals T9orTi2.
a Avg. ACt and SD based on replicate swatches 1 and 2; the 3rd swatch replicate had higher Ct values (~26 at T9) and was not
included in the average and SD or pooled SD.
b Avg. ACt and SD based on replicate swatches 2 and 3; the 1st swatch replicate had higher Ct values, ~27 and ~33 at T9 and
T12, respectively, and was not included in the average and SD or pooled SD.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. = Average; SD =
Standard Deviation; ND = Non-Detect; Reps = Replicates.
Together these results for swab swatches processed by vortexing in PBS followed by UF-concentration
(~20-fold reduction by volume), using 2-hr infection and T9 incubation in Vero E6 cell culture, showed
consistent detection of low SARS-CoV-2 levels. RV-RVPCR results showed 3 of 3 positive replicates
for ~21 PFU/well (avg. ACt -9.8-11.5). However, for swatches with -17 PFU/well and -15 PFU/well,
2 of 3 replicates were positive with avg. ACt values of-7.1-9.8 and -9.3-10.6, respectively.
55

-------
4.4.4 Comparison of RV-RT-PCR and TCID50 analyses from the same SARS-CoV-2-
spiked swab swatch samples (side-by-side analysis)
An experiment was conducted using the same swab swatch for both RV-RT-PCR and TCID50 analysis
instead of using separate swabs swatches. Like the previous swab swatch experiments, 0.5 mL from
viral dilutions -3 and -3.3 logio were used to spike swatches in triplicate, and triplicate negative controls
were included. After swatch processing, 0.5 mL viral UF-retentate was obtained; however, in this case,
0.1 mL were added to To, T9, and T12 cell culture plates and 0.1 mL was also used for TCID50 analysis.
Based on the TCID50 analysis of the viral stock used for this experiment (TCID50 10517/0.1 mL or
-1.03 x 105 PFU/0.1 mL), -514 and -257 PFU were added per swatch for the -3 and -3.3 logio dilutions,
respectively. This experiment was referred to as "side-by-side RV-RT-PCR and TCID50 analyses,"
although the swatch UF-retentate was not split in half for both analyses since only 0.1 mL was needed to
make an initial 10-fold dilution per the standard TCID50 method.
The viral percent recovery results are shown in Table 38. The "Estimated Starting PFU/Swatch" was
calculated from the TCID50 of the virus stock and the PFU for RV-RT-PCR analysis was calculated
from the "side-by-side" TCID50 analysis from the same swatch. For starting PFU/swatch levels of-600
and -300, the average percent recovery values were -42.7% and -38.2%, respectively. For actual
sample analysis, the recovered swatch UF-retentate (-0.2 mL) will be divided in two parts, 0.1 mL for
To and 0.1 mL for T9. Therefore, the PFU for RV-RT-PCR analysis (in the table) represents the PFU per
0.2 mL that would be split in half for To and T9 analyses. The average PFU for RV-RT-PCR was -88
and -39 PFU for starting PFU/swatch levels of -514 and -257, respectively.
Table 38 Recovery Efficiency of SARS-CoV-2 from Swab Swatches
Ksliiiiiilod
Sl:irlin»
PI 1 /
Swjilch"
Swiilch
Rcplicnlc
Reco\ iroil
PI I /
SwnU'h
Percent
Reco\cry
(%)
PI I lor RV-RT-
PCR
Analysis	

1
111
21.5
44.3
514
2
350
68.1
140.0
3
197
38.3
78.7

A\«. (SI))
219(121)
42.7 (23.(>)
87.7 (48.5)

1
197
76.6
78.7
257
2
35
13.6
14.0
3
62
24.2
24.9

\\». (SI))
98 (87)
38.2 (33.7)
3<).2 (34.7)
* Values are based on dilutions from a SARS-CoY-"' uicivd slock (ICI 1)^0 = 10517/0.1 mL or
1.03 x 105 PFU/0.1 mL), using 0.5 mL of either -3 or -3.3 log 10 dilution per swab swatch.
TCID50/0.1 mL with SD = 0.99-2.17 x 105 and PFU/0.1 mL with SD = 0.69-1.52 x 105.
** Values are based on TCID50 analysis of UF-retentates from the same swatch used for RV-RT-
PCR analysis.
*** Values are based on TCID50 analysis of swatch UF-retentates using 0.2 mL as the total volume
since 0.1 mL was used for T0 and 0.1 mL was used T9 for RV-RT-PCR analysis.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Avg. = Average; SD
= Standard Deviation.
The RV-RT-PCR results for this experiment are shown in Table 39, with the corresponding "PFU for
RV-RT-PCR Analysis" data included for each swatch replicate. For both starting viral levels (-257 and
-514 PFU/swatch), 3 of 3 swatch replicates showed positive results with ACt values ranging from 9.8 to
56

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11.2 for the -514 PFU level (-88 PFU for RV-RT-PCR analysis) and from 7.6 to 11.2 for the -257 PFU
level (-39 PFU for RV-RT-PCR analysis). Results for the negative control were non-detect (data not
shown). The RV-RT-PCR results are also summarized for Avg. Ct and Avg. ACt values in Table 40.
The T12 incubation period was included to provide data if the T9 incubation did not result in detection of
3 of 3 swatches with ACt > 6 for these lower viral levels. However, T12 wells were not processed or
analyzed since T9 results met the requirement for ACt > 6 for all three swatch replicates.
Table 39 Side-by-Side TCID50 and RV-RT-PCR Results for SARS-CoV-2-Spiked Swab
Swatches Processed and Used to Infect Vero E6 Cells with 2-hr Infection
Ksti 111:1 (oil
PI-1 /
Swnlch
Switch
Rcpliciilc
PI 1 lor
RV-RT-
PCR
RT-PCR
Rcpliciitc
Ci hy Posl-lnlection
Inciihiition Timcpoinl
Alter 2-hr Inloction	
A\g. AC 'i
(SI) )'•'¦•'¦•'•

An:ilvsis" -

In
1,




1
32.4
21.4


1
44.3
2
32.7
21.3
11.2

3
32.3
21.3
(0.2)



Avg. (SD)
32.5 (0.2)
21.3 (0.1)




1
32.1
22.4


2
140
2
32.1
22.5
9.8
514
3
32.4
22.4
(0.1)


Avg. (SD)
32.2 (0.1)
22.4 (0.04)




1
31.8
21.3


3
78.7
2
31.9
21.2
10.5

3
31.8
21.4
(0.1)



Avg. (SD)
31.8 (0.1)
21.3 (0.1)


A\«. (SI))
S7.7 (4S.5)
Om'IJIII
A>g. (SI))
32.2 (0.3)
21.7 (0.6)
10.5 (0.6)



1
33.2
25.5


1
78.7
2
32.9
25.5
7.6

3
33.1
25.5
(0.1)



Avg. (SD)
33.1 (0.2)
25.5 (0.04)




1
33.7
22.4


2
14.0
2
33.8
22.5
11.2
257
3
33.4
22.5
(0.1)


Avg. (SD)
33.6 (0.2)
22.5 (0.03)




1
33.4
24.0


3
24.9
2
33.4
23.8
9.4

3
33.0
23.9
(0.2)



Avg. (SD)
33.3 (0.2)
23.9 (0.1)


Ay... (SI))
39.2 (34.7)
(ho 1:1 II
A\ g. (SI))
33.3 (0.3)
24.0(1.3)
9.4(1.6)
* Values arc based 011 dilutions 1'roni a SARS-Co\'-2 lileied siock (TCM)N> in n.l 111L or 1.03 lu H I I/O. 1
mL). Estimated PFU/Swatch = 0.7 x TCID50/Swatch.
** Values are based on TCID50 analysis of swatch UF-retentates using 0.2 mL as the total volume since 0.1 mL
was used for T0 and 0.1 mL was used T9 for RV-RT-PCR analysis (Table 38).
*** Avg. and SD are from triplicate RT-PCR analyses per swatch replicate.
**** sd represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus
the SD for the T9 values squared)/2.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. =
Average; SD = Standard Deviation.
57

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Table 40 Summary of Side-by-Side TCID50 and RV-RT-PCR Analyses for SARS-CoV-2-Spiked
Swab Swatches Processed and Used to Infect Vero E6 Cells with 2-hr Infection
Ksliniiileil
Stsi rtin«>
PI-1 /
Swiilch"
Swsilch
Repliciile
PI I lor RV-
RT-PCR
Aiiiilvsis'" -
A\». C'i (SI)) by Post-
In led ion Inciihiilion
Timepoinl
Alter 2-hr Inledion	
A\». AC 'i
(SI))1-"


In
T,


1
44
3:.5 (UJ)
21.3 (U.l)
11.2 (U.2)
514
2
140
32.2 (0.1)
22.4 (0.04)
9.8 (0.1)
3
79
31.8 (0.1)
21.3 (0.1)
10.5 (0.1)

\\». (SI))
SS (49)
32.2 (0.3)
21.7 (0.6)
10.5 (0.6)

1
7y
33 1 (() 2)
25.5 (() i)4)
7.6 (0.1)
257
2
14
33.6(0.2)
22.5 (0.03)
11.2 (0.1)
3
25
33.3 (0.2)
23.9 (0.1)
9.4 (0.2)

.^«.(SD)
39 (35)
33.3 (0.3)
24.0(1.3)
9.4(1.ft)
* Values are based on dilutions from a SARS-CoV-2 titered stock (TC1D5U lu 0.1111L or 1.03 lu
PFU/0.1 mL). Estimated PFU/Swatch = 0.7 x TCID50/Swatch.
** Values are based on TCID50 analysis of swatch UF-retentates using 0.2 mL as the total volume since 0.1
ml. was used for T0 and 0.1 mL was used T9 for RV-RT-PCR analysis (Table 38).
*** Avg. and SD are from triplicate RT-PCR analyses per swatch replicate.
**** represents the pooled SD, which equals the square root of the following: (SD for T0 values squared
plus the SD for the T9 values squared)/2.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg.
= Average; SD = Standard Deviation.
A replicate "side-by-side RV-RT-PCR and TCID50 analyses" experiment was conducted using the same
swab swatch for both RV-RT-PCR and TCID50 analysis (side-by-side analyses). As described
previously, 0.5 mL from viral dilutions "3 and -3.3 logio were used to spike swab swatches in triplicate,
and triplicate negative controls were included. After swatch processing to recover virus, 0.5 mL UF-
retentate was obtained and aliquots of 0.1 mL were added to To, T9, and T12 cell culture plates with 0.1
mL used for TCID50 analysis. Replicate swab swatches were included to determine viral recovery
efficiency via TCID50 analysis, using the same viral dilutions, -3 and -3.3 logio. Based on the TCID50
analysis of the viral stock used for this experiment, -134 and -67 PFU were added per swatch based on
dilutions of the titered stock.
The viral percent recovery results are shown in Table 41. For starting PFU/swatch levels of-134 and
-67, the average percent recovery values were -46.4% and -40.8%, respectively. The recovery
efficiency values > 100% were not included in the calculation of average percent recovery (for 67 and
134 PFU/swatch, one replicate for each viral level), because these values were inconsistent with the
other swatch replicates, and likely represent technical error in swatch set-up or processing. The average
PFU for RV-RT-PCR analysis (PFU/0.2 mL with 0.1 mL for To and 0.1 mL for Tf) was -25 and -11
PFU for starting PFU levels of-134 and -67, respectively.
58

-------
Table 41 Recovery Efficiency of SARS-CoV-2 from Swab Swatches - Replicate Experiment
Ksliniiiled
Sliirliii" PI-1 /
Swsilch"
Swsilch
Rcplic;ilc
Reco\ ereil
PI I /
Swiilch
Percent
Recoxcry
(%)
PI I lorRY-RT-PC R
A n;i lysis
134
1
62.2
46.4
24.9
2
>134
>100
>55.6
3
62 2
46 4
24 9
A\«. (SI))
62.2 (0)
46.4 (0)
24/) (0)
67
1
35.0
52.2
14.0
2
>67
>100
>28
3
197
29 4
7.9
A\«. (SI))
27.3(10.8)
40.8 (16.2)
10.9 (4.3)
* Values are based on dilutions from a SARS-CoV-2 titered stock (TCID50 104 6/0.1 mL or 2.68 x 104
PFU/0.1 mL), using 0.5 mL of either -3 or -3.3 logio dilution per swab swatch. TCID50/0.1 mL with SD =
3.2-4.6 x 104 and PFU/0.1 mL with SD = 2.2-3.25 x 104.
** Based on TCID50 analysis of swatch UF-retentates from sample processing. TCID50 values showing
>100% recovery were not included in calculation of the average recovery (see text for explanation).
*** Values are based on TCID50 analysis of swatch UF-retentates using 0.2 mL as the total volume since
0.1 mL was used for T0 and 0.1 mL was used T9 for RV-RT-PCR analysis.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg.
= Average; SD = Standard Deviation.
Results for RV-RT-PCR analysis of swab swatches spiked with these viral levels are shown in Table 42.
For -134 PFU/swatch (-25 PFU for RV-PTPCR analysis), 3 of 3 replicates were detected with average
ACt of 8.1, however, for the lower viral level of-67 PFU/swatch (-11 PFU for RV-PTPCR analysis), 2
of 3 replicates were detected with an average ACt of 9.8. Results for the negative control swatches were
non-detect (data not shown). Since 2 of 3 T9 swatch replicates showed positive ACt values (8.3-11.4)
for the -67 PFU/swatch level, T12 wells were not analyzed.
59

-------
Table 42 Side-by-Side TCID50 and RV-RT-PCR Results for SARS-CoV-2-Spiked Swab Swatches
Processed and Used to Infect Vero E6 Cells with 2-hr Infection - Replicate Experiment
Ksliniiitcd
PI I /
S\\;i U'h
Swsitch
Rcplic;itc
PI I /
Swsilch for
RV-RT-PCR
RT-PCR
Rcpliciitc
Ci hv Post-1 nI'oclion 1 ncnh:ilion
Timcpoint Al'lcr 2-hr
In lection---
A\». ACi
(SI))-

An silvsis- -

In
1,




1
32.9
23.1


1
24.9
2
32.7
23.2
9.7 (0.09)

3
33.0
23.1



Avg. (SD)
32.9 (0.1)
23.1 (0.04)




1
34.1
26.3


2
(>55.6)
2
33.5
26.3
7.6(0.3)
134
3
34.3
26.4


Avg. (SD)
34.0 (0.4)
26.3 (0.1)




1
33.4
26.7


3
24.9
2
33.1
26.6
6.8 (0.3)

3
33.8
26.6



Avg. (SD)
33.5 (0.4)
26.6 (0.02)


An«. (SI))
24.9 (0)
OmT2|II A\».
(SI))
33.4 (0.5)
25.4(1.7)
8.1 (1.8)



1
35.1
26.8


1
14.0
2
35.2
26.7
8.3 (0.1)

3
35.0
26.9



Avg. (SD)
35.1 (0.1)
26.8 (0.1)




1
35.4
23.7


2
(>28)
2
35.4
23.8
11.4 (0.3)

3
34.6
23.7
67


Avg. (SD)
35.1 (0.5)
23.7 (0.02)




1
35.5
35.9


3
7.9
2
35.6
36.2
-0.5 (0.2)

3
35.4
35.8



Avg. (SD)
35.5 (0.1)
36.0 (0.2)


A\». (SI))
10/) (4.3)
Omt;iII A\».
(SI)) (3 Reps)
35.2 (0.3)
2S.S (5.5)
6.4 (5.3)

OMTilll A\».
(SI)) (2 Reps)
35.1 (0.3)
25.3(1.7)
9.8(1.7)
* Values are based on dilutions from a SARS-CoV-2 titered stock (TCID50 104 6/0.1 mL or 2.68 x 104 PFU/0.1 mL).
Estimated PFU/Swatch = 0.7 x TCID50/Swatch.
** Values are based on TCID50 analysis of swatch UF-retentates using 0.2 mL as the total volume since 0.1 mL was used for
T0 and 0.1 mL was used T9 for RV-RT-PCR analysis (Table 41). TCID50 values showing >100% recovery were not
included in calculation of the average recovery (see text for explanation).
60

-------
*** Avg. and SD are from triplicate RT-PCR analyses per swatch replicate. The Overall Avg. and SD are based
on three replicate swatches unless specified as 2 or 3 replicates.
**** represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the T9 values squared)/2.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. = Average; SD =
Standard Deviation; Reps = Replicates.
Following the same swatch processing conditions described previously, these experiments for swab
swatches analyzed by RV-RT-PCR and TCID50 using the same swatch ("side-by-side" analyses)
showed 3 of 3 positive swatches for -134 PFU/swatch by RV-RT-PCR (using 2-hr infection and T9
incubation), with average ACt values ranging from 6.8-9.7. Since only about half the swatch UF-
retentate (0.2 mL of 0.5 mL) was used for RV-RT-PCR analysis (0.1 mL each for To and T9), the results
suggested that -67 PFU/swatch would yield similar results if the whole swatch UF-retentate was used
for analysis. When -67 PFU/swatch was tested in this side-by-side analysis format (with an estimated
33 PFU/swatch used for RV-RT-PCR analysis), 2 of 3 replicates were positive for RV-RT-PCR with
average ACt values of 8.3 and 11.4. The third replicate had the lowest recovery efficiency with only
-20 PFU recovered after processing. Therefore, the appropriate To and T9 cell culture wells each had -4
PFU/0.1 mL added, which was not detected. Based on this analysis, the method sensitivity of detection
was estimated to be <100 virions per swatch), although this needed to be confirmed with swabs using
the whole UF-retentate analyzed by the optimized RV-RT-PCR method as described below. These
results suggested if the entire swatch UF-retentate were processed for RV-RT-PCR analysis, estimated
starting PFU/swatch levels of-67 or lower could be reliably detected by RV-RT-PCR analysis.
In summary, the optimized RV-RT-PCR parameters and conditions included 96-well Vero E6 cell
culture (seeded with 35,000 cells/well), swatch processing in 10-mL PBS by vortex-mixing, followed by
concentration using ultrafiltration (with up to -50-fold volume reduction if 10-mL is concentrated to
0.2-mL), and 0.22-micron filtration to remove microbial contaminants. Further, RV-RT-PCR analysis
included splitting the recovered swatch UF-retentate (-0.2 mL) in half, with -0.1 mL each added to two
96-well Vero E6 cell culture plates (for To and T9), along with -0.1 mL cell culture medium, since UF-
retentates were in PBS. After a 2-hr infection period, and washing the plate wells to remove free virus,
the To plate was prepared for subsequent RNA extraction. The T9 plate was incubated for 9 hr, and
subsequently prepared for RNA extraction. The overall method, which uses a magnetic bead-based
RNA extraction method followed by RT-PCR analysis, showed an estimated sensitivity of detection for
swab swatches between -67 and -33 PFU/swatch based on "side-by-side" RV-RT-PCR/TCID50
analysis as described above, if the entire swatch UF-retentate was used for RV-RT-PCR analysis. The
TCID50 analysis of swatch UF-retentates showed that for estimated -14 PFU for RV-RT-PCR analysis
(-14 PFU in 0.2 mL with 0.1 mL each used for To and T9), average ACt values were 8.3 and 11.2
(shown in Table 42 and Table 39, respectively), well above the ACt > 6 requirement for positive
detection.
4.4.5 Analysis of SARS-CoV-2-spiked swab samples using the final optimized RV-RT-
PCR method
Using the optimized RV-RT-PCR method as described above, actual swabs were tested with three
SARS-CoV-2 dilutions including -3, -3.3, and -4 logio relative to the virus stock (TCID50 1 05 36/0.1 mL
or -1.6 x 105 PFU/0.1 mL), along with a negative control (PBS added to swabs), using triplicate swabs
per viral level. Swabs were obtained from Sanigen (Sani-MacroSwab, Anyang, Gyeonggi-do, South
Korea). The swab tubes contained 10 mL PBS, therefore, 0.5 mL liquid was removed to accommodate
spiking with 0.5 mL SARS-CoV-2 dilution to the swab head. Liquid was expressed from the swab
before adding the viral dilution. Swabs were processed as described for swab swatches; however, after
61

-------
vortex mixing the tube, the recovered swab suspension (with virus) was concentrated by UF to -0.2 mL
instead of 0.5 mL and split in half between the appropriate cell-culture wells on To and T9 plates. As
described previously, 0.1 mL cell culture medium was then added to these wells, and infection was
conducted for 2 hr, followed by T9 incubation. In addition, recovery efficiency was determined by
conducting TCID50 analysis of replicate swabs spiked with 10"2 or 10"3 viral dilutions in triplicate.
The viral percent recovery results are shown in Table 43. For starting PFU/swab levels of-6,200 and
-620, the average percent recovery values were -54% and -35%, respectively. Based on the average of
these percent recovery values (44.8%), the average PFU for RV-RT-PCR analysis (PFU/0.2 mL as
described above) were estimated to be -220, -139, and -28 for starting PFU/swab levels of 620, 310,
and 62, respectively.
Table 43 Recovery Efficiency of SARS-CoV-2 from Swabs
Ksliniiiled
Sl;irlin» PR /
Swiib"
Swiib
Repliciile
Recox ereil
PI-1 /
Sw it h
Percent Reco\erv
(%)
Kstiniiited A\». PR
lor RV-RT-PCR
Analysis	

1
6200
100

6200
2
1970
31.6
NA
3
1970
31.6

A\«. (SI))
334>0 (2460)
54(40)


1
350
56.2

620
2
111
17.8
-220
3
197
31.6

A\». (SI))
21') (121)
35(20)


A\». Percent Rcco\crv
44.S

310

Not
Determined
Not Determined
-139
62

Not
Determined
Not Determined
-28
* Values are based on dilutions from the SARS-CoV-2 titered stock (TCID50 105 25/0.1 mL or 1.24 x 105 PFU/0.1
mL), using 0.5 mL of either 10"2 or 10"3 dilution per swab, 6200 or 620 PFU, respectively. TCID50/0.1 mL with
SD = 1.32-2.40 x 105 and PFU/0.1 mL with SD = 0.92-1.68 x 105.
** Values are based on TCID50 analysis of UF-retentates from replicate swabs, where PFU/Swab = TCID50/Swab x
0.7. Avg. and SD of three PFU/swab values for each viral dilution tested.
*** Values are based on actual measurement of recovered virus by TCID50 analysis for swabs spiked with 6,200
and 620 PFU and calculated for swabs spiked with 310 or 62 PFU using an average recovery efficiency
(determined from the other virus dilutions). Recovered PFU in ~0.2 mL were split between To and T9 wells.
NA = Not Applicable; This viral dilution was not used to spike swabs.
Not Determined; Recovery efficiency was not determined directly from these viral dilutions.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Avg. = Average; SD = Standard
Deviation.
RV-RT-PCR results using the N1 assay for the first experiment are shown in Table 44 for the three viral
dilutions. The negative control was non-detect for all three replicates (data not shown). The estimated
PFU/swab based on TCID50 analysis of the viral stock (-1.24 x 105 PFU/0.1 mL) were approximately
620, 310, and 62 for the three different viral dilutions. The estimated PFU per swab based on recovery
efficiency are also included in the table. The data showed positive RV-RT-PCR results for 3 of 3 swab
replicates after 2-hr infection and 9 hr (T9) post-infection incubation for all three starting viral levels.
62

-------
Table 44 RV-RT-PCR Results (N1 Assay) for SARS-CoV-2-Spiked Swabs Processed and
Used to Infect Vero E6 Cells with 2-hr Infection
KslimaU'd
Starling
PI-1 /
KslimaU'd
A\«. 1*1" 1
Ucco\ crcd
Swab
Ucplicalc
RT-I'CR
Ucplicalc
C'i hy I'osl-lnlcclion
1 ncu hat ion Timcpoinl
Alter 2-hr In lection'""'"'
A\g. AC 'i
(SI))-
Sw ;i h


I",,
1,




1
31.0
20.7



1
2
30.9
20.8
10.2 (0.1)


3
31.1
20.9



Avg. (SD)
31.0 (0.1)
20.8 (0.1)




1
30.7
19.0



2
2
30.6
19.2
11.6 (0.1)
620
220
3
30.6
19.0



Avg. (SD)
30.7 (0.1)
19.0 (0.1)




1
31.4
20.5



3
2
31.3
20.3
11.1 (0.2)


3
31.3
19.9



Avg. (SD)
31.4 (0.1)
20.2 (0.3)



(>\erall
\\«.(SI»
31.0(0.3)
20.0 (0.S)
11.0(0.6)



1
32.1
21.1



1
2
31.9
21.1
11.0 (0.1)


3
32.1
21.0



Avg. (SD)
32.0 (0.2)
21.0 (0.1)




1
32.3
21.5



2
2
32.4
21.0
10.9 (0.2)
310
139
3
31.9
21.5



Avg. (SD)
32.2 (0.2)
21.3 (0.3)




1
31.8
21.0



3
2
31.8
20.9
10.9 (0.1)


3
31.9
20.9



Avg. (SD)
31.9 (0.1)
21.0 (0.08)


(he rail
\>g. (SI))
32.0 (0.2)
21.1 (0.2)
10.9 (0.2)
* Values are based on dilutions from a SARS-CAA -2 lileivd slock (TCII)N) lu u.lniLorU4 lu Pl'l U.l inL).
Estimated PFU/Swab = 0.7 x TCID50/Swab.
** Avg. values are based on SARS-CoV-2 recovery determined from replicate swabs (Table 43).
*** Avg. and SD are from triplicate RT-PCR analyses per swab replicate. The Overall Avg. and SD are based
on three replicate swabs.
**** represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the T9 values squared)/2.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. =
Average; SD = Standard Deviation.
63

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Table 44 RV-RT-PCR Results (N1 Assay) for SARS-CoV-2-Spiked Swabs Processed and
Used to Infect Vero E6 Cells with 2-hr Infection (Cont'd)
Ksliimitcd
Stirling
PI-1 /
Ksliiiiiilod
A\«. 1*11
Rcco\ civil • •
Swjib
Rcplicittc
R1-PC R
Rcplicitlc
C'i hv Post-In lection
1 lieu bill ion Timcpoinl
Afler 2-hr liilci'lion
A\g. A( 'i
(SI))-• •• ••
Swjib"


I",,
1,




1
35.4
22.2



1
2
35.1
22.2
13.4 (0.4)


3
36.1
22.2



Avg. (SD)
35.5 (0.5)
22.2 (0.03)




1
35.4
22.5



2
2
35.2
22.5
12.5 (0.3)
62
28
3
34.4
22.6



Avg. (SD)
35.0 (0.5)
22.5 (0.1)




1
35.2
23.3



3
2
36.1
23.2
12.2 (0.4)


3
35.0
23.2



A\g. (SI))
35.4 (0.6)
23.2 (0.04)



OmtsiII .
V\g. (SI))
35.3 (0.5)
22.7 (0.5)
12.7(0.6)
* Values are based on dilutions from a SARS-CoV-2 titered stock (TCID50 105 25/0.1 mL or 1.24 x 105 PFU/0.1 mL).
Estimated PFU/Swab = 0.7 x TCID50/Swab.
** Avg. values are based on SARS-CoV-2 recovery determined from replicate swabs (Table 43).
*** Avg. and SD are from triplicate RT-PCR analyses per swab replicate. The Overall Avg. and SD are based
on three replicate swabs.
**** represents the pooled SD, which equals the square root of the following: (SD for To values squared plus the SD for
the T9 values squared)/2.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. = Average; SD
= Standard Deviation.
Average ACt values were 11.0 ± 0.6, 10.9 ± 0.2, and 12.7 ± 0.6 for -620, -310, and -62 estimated
PFU/swab, respectively. RV-RT-PCR results using the N2 assay also showed 3 of 3 swab replicates
positive for all three virus levels with average ACt values of 11.5 ± 0.6, 11.2 ± 0.2, and 13.1 ± 0.5 for
-620, -310, and -62 estimated PFU/swab, respectively (Table 45). The negative control swabs were
also non-detect for the N2 assay for all three replicates (data not shown). The data showed a less than
100-virion) sensitivity of detection could be achieved with RV-RT-PCR analyses using the N1 and N2
assays.
64

-------
Table 45 RV-RT-PCR Results (N2 Assay) for SARS-CoV-2-Spiked Swabs Processed and
Used to Infect Vero E6 Cells with 2-hr Infection
Kstiimitcil
Slii rlin»
PI-1 /
Sw ;i h
Ksliiiiiiloil
A\«. 1*11 /
Swsih
lor RY-RT-
I'C'R
Annlvsis•
Swjib
Repliciilo
RT-I'CR
Ropliciito
C'i hv I'osl-lnlcclion
Inciihiilion limopoint
Alter 2-hr Infection"""
A\«. AC i
(SI))-
In
T..
620
220
1
1
30.1
19.4
10.7 (0.1)
2
30.2
19.4
3
30.2
19.5
Avg.
30.1 (0.1)
19.4 (0.1)
2
1
29.9
17.8
12.0 (0.1)
2
29.9
17.9
3
29.8
17.8
Avg.
29.9 (0.1)
17.8 (0.04)
3
1
31.0
19.2
11.7 (0.2)
2
30.7
19.2
3
30.8
18.9
Avg.
30.8 (0.2)
19.1 (0.2)
OmtsiII A\». (SI))
30.3 (0.4)
18.8(0.7)
11.5 (0.6)
310
139
1
1
31.3
19.9
11.3 (0.2)
2
31.3
20.1
3
31.1
19.7
Avg.
31.2 (0.1)
19.9 (0.2)
2
1
31.6
20.7
11.0 (0.1)
2
31.6
20.5
3
31.7
20.6
Avg.
31.6 (0.04)
20.6 (0.1)
3
1
31.2
19.8
11.4 (0.1)
2
31.1
19.7
3
31.2
19.7
Avg.
31.1 (0.1)
19.8 (0.05)
Owi'hII A\».(SD) 31.3(0.2) 20.1(0.4) 11.2(0.2)
* Values are based on dilutions from a SARS-CoV-2 titered stock (TCID50 105 25/0.1 mL or 1.24 x 105 PI "I H i mL).
Estimated PFU/Swab = 0.7 x TCID50/Swab.
** Avg. values are based on SARS-CoV-2 recovery determined from replicate swabs (Table 43).
*** Avg. and SD are from triplicate RT-PCR analyses per swab replicate. The Overall Avg. and SD are based
on three replicate swabs.
**** represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the T9 values squared)/2.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. =
Average; SD = Standard Deviation.
65

-------
Table 45 RV-RT-PCR Results (N2 Assay) for SARS-CoV-2-Spiked Swabs Processed and
Used to Infect Vero E6 Cells with 2-hr Infection (Cont'd)
KslimiiU'd
PI-1 /
Sw ;i h
Kst i ill ;i toil
A\». I'l'l lor
RY-RT-IH R
Ansilvsis"-
Swjib
Rcplicitlc
RT-I'CR
Rcplicittc
C'i hv Post-Inloction
1 n cu hilt ion l i inopoi nt After
2-hr Infection-
A\». A( 'i
(SI))-• •• ••
T„
1,
62
28
1
1
34.6
21.1
13.7
(0.3)
2
34.3
20.9
3
35.2
21.0
Avg. (SD)
34.7 (0.4)
21.0 (0.1)
2
1
34.3
21.3
12.9
(0.1)
2
34.0
21.2
3
34.2
21.3
Avg. (SD)
34.2 (0.2)
21.3 (0.1)
3
1
34.8
22.4
13.0
(0.4)
2
35.9
22.4
3
35.5
22.3
Avg. (SD)
35.4 (0.6)
22.4 (0.1)
OmtsiII A\». (SI))
34.7 (0.7)
21.6 (0.6)
13.1 (0.5)
* Values are based on dilutions from a SARS-CoV-2 titered stock (TCID50 105 25/0.1 mL or 1.24 x 105 PFU/0.1 mL).
Estimated PFU/Swab = 0.7 x TCID50/Swab.
** Avg. values are based on SARS-CoV-2 recovery determined from replicate swabs (Table 43).
*** Avg. and SD are from triplicate RT-PCR analyses per swab replicate. The Overall Avg. and SD are based
on three replicate swabs.
**** represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the T9 values squared)/2.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. =
Average; SD = Standard Deviation.
The average Ct and average ACt results for triplicate swabs (with triplicate RT-PCR analyses) from
Table 44 (N1 assay) and Table 45 (N2 assay) are summarized in Table 46 and Table 47, respectively.
66

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Table 46 Summary of RV-RT-PCR Avg. Ct and ACt Results for SARS-CoV-2-Spiked Swabs
Processed and Used to Infect Vero E6 Cells with 2-hr Infection - N1 Assay
Ksliiiiiilod
Sljirliii" Pi t /
SwjiIv-
Kstiimilcd
A\». I'l'l lor
RY-RT-I'CR
S\\:ib
Replicitle
A\». C'i hv I'osl-lnlcclion
1 iicnhiition l imopoilit
Allir 2-hr 1 n loci ion	
A\j>. AC i
(SI))-""-
Ansilvsis--

In
1 ..



1
31.0 (0.1)
20.8 (0.1)
10.2 (0.1)
620
220
2
30.7 (0.1)
19.0(0.1)
11.6(0.1)
3
31.4 (0.1)
20.2 (0.3)
11.1 (0.2)


A>«>. (SI))
31.0 (0.3)
20.0 (O.S)
1 1.0(0.6)


1
32.0 (0.2)
21.0(0.1)
11.0(0.1)
310
139
2
32.2 (0.2)
21.3 (0.3)
10.9(0.2)
3
31.9 (0.1)
21.0 (0.08)
10.9(0.1)


A\s.(SI»
32.0 (0.2)
21.1 (0.2)
10.') (0.2)


1
35.5 (0.5)
::: (t> d3>
13 4(0 4)
62
28
2
35.0 (0.5)
22.5 (0.1)
12.5 (0.3)
3
35.4 (0.6)
23.2 (0.04)
12.2 (0.4)


Ay... (SI))
35.3 (0.5)
22.7 (0.5)
12.7 (0.(>)
* Values are based on dilutions from a SARS-CoV-2 titered stock (TCID50 105 25/0.1 mL or 1.24 x 105 PFU/0.1 mL).
Estimated PFU/Swab = 0.7 x TCID50/Swab.
** Avg. values are based on SARS-CoV-2 recovery determined from replicate swabs (Table 43).
*** Avg. and SD are from triplicate RT-PCR analyses per swab replicate.
**** represents the pooled SD, which equals the square root of the following: (SD for To values squared plus the SD for
the T9 values squared)/2.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. =
Average; SD = Standard Deviation.
67

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Table 47 Summary of RV-RT-PCR Avg. Ct and ACt Results for SARS-CoV-2-Spiked Swabs
Processed and Used to Infect Vero E6 Cells with 2-hr Infection - N2 Assay
Estimated
Starting
PFU /
Swab*
Estimated
Avg. PFU for
RV-RT-PCR
Analysis**
Swab
Replicate
Ct by Post-Infection
Incubation Timepoint
After 2-hr Infection***
Avg. ACt
To
T9
620
220
1
30.1 (0.1)
19.4 (0.1)
10.7(0.1)
2
29.9 (0.1)
17.8 (0.04)
12.0(0.1)
3
30.8 (0.2)
19.1 (0.2)
11.7(0.2)
Avg. (SD)
30.3 (0.4)
18.8 (0.7)
11.5 (0.6)
310
139
1
31.2 (0.1)
19.9(0.2)
11.3 (0.2)
2
31.6(0.04)
20.6(0.1)
11.0(0.1)
3
31.1 (0.1)
19.8 (0.05)
11.4 (0.1)
Avg. (SD)
31.3 (0.2)
20.1 (0.4)
11.2 (0.2)
62
28
1
34.7 (0.4)
21.0(0.1)
13.7(0.3)
2
34.2 (0.2)
21.3 (0.1)
12.9(0.1)
3
35.4 (0.6)
22.4 (0.1)
13.0(0.4)
Avg. (SD)
34.7 (0.7)
21.6 (0.6)
13.1 (0.5)
* Values are based on dilutions from a SARS-CoV-2 titered stock (TCID50 105 25/0.1 inL or 1.24 x 105 PFU/0.1 inL).
Estimated PFU/Swab = 0.7 x TCID50/Swab.
** Avg. values are based on SARS-CoV-2 recovery determined from replicate swabs (Table 43).
*** Avg. and SD are from triplicate RT-PCR analyses per swab replicate.
**** represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the T9 values squared)/2.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. =
Average; SD = Standard Deviation.
A replicate experiment was conducted with Sanigen swabs using the optimized RV-RT-PCR method
(including 2-hr infection and 9-hr incubation) as described above. Swabs were prepared as described
previously, however, in this case slightly lower viral levels were used. Triplicate swabs were spiked
with viral dilutions -3.3, -4 and -4.3 logio relative to the virus stock (in addition to triplicate negative
control swabs). For this experiment, the viral titer was somewhat lower, namely TCID50 105 °/0.1 mL
or ~7 x 104 PFU/0.1 mL for this experiment compared to TCID50 1 05 36/0.1 mL or-1.6 x 105 PFU/0.1
mL for the first experiment. In this case, recovery efficiency was also determined by TCID50 analysis
of replicate swabs spiked with 10"2 or 10"3 viral dilutions in triplicate.
The viral percent recovery results are shown in Table 48. For starting PFU/swab levels of-3500 and
-350, the average percent recovery values were -78% and -44%, respectively. Based on the average of
these percent recovery values (61%), the average PFU for RV-RT-PCR analysis were estimated to be
-107, -21, and -11 PFU for starting PFU/swab levels of-175, -35, and -18, respectively.
RV-RT-PCR results using the N1 assay for the first experiment are shown in Table 49 for the three viral
dilutions. The negative control was non-detect for all three replicates (data not shown). The estimated
PFU/swab based on TCID50 analysis of the viral stock were approximately 175, 35, and 18 for the three
different viral dilutions based on the titer of the SARS-CoV-2 stock (avg. 7.0 x 104 PFU/0.1 mL), and
the estimated PFU per swab based on recovery efficiency are also included in Table 49. The data
showed positive RV-RT-PCR results for 3 of 3 swab replicates after 2-hr infection and 9 hr post-
68

-------
infection incubation two of the three starting viral levels, showing ACt values > 6 for -175 and -35
PFU/swab levels, although the lowest virus level (-18 PFU/swab) was non-detect for all three swab
replicates. The results were similar for the N2 assay shown in Table 50. The negative control swabs
were also non-detect for the N2 assay for all three replicates (data not shown). Average ACt values
were 11.4 and 11.8 for the N1 and N2 assays, respectively for -175 PFU/swab and 13.3 and 14.1 for the
N1 andN2 assays, respectively for -35 PFU/swab.
The average Ct and average ACt results for triplicate swabs (with triplicate RT-PCR analyses) from
Table 49 (N1 assay) and Table 50 (N2 assay) are summarized in Table 51 and Table 52, respectively.
Table 48 Recovery Efficiency of SARS-CoV-2 from Swabs - Replicate Experiment
Ksliniiilcd
Sliirtin<> PR /
S \\;ih •
Swiib
Repliciile
Recox ereil
PI I /
Sw ;i h •
Percent
Reco\ erv
(%)
Kstiniiited A\». PR
lor R\ -RT-PCR
Ansilvsis" --
3500
1
1970
56
NA
2
3500
100
3
>3500
>100
A\«. (SI))
1300 (»)52)
78(31)
350
1
197
56
NA
2
111
32
3
>350
>100
Ay... (SI))
154 ((>1)
44(17)

A\». Percent Reco\erv
61

175

Not
Determined
Not
Determined
107
35

Not
Determined
Not
Determined
21
18

Not
Determined
Not
Determined
11
* Values are based on dilutions from a SARS-CoV-2 titered stock (TCID50 105 °/0.1 mL or 7.0 x 104 PFU/0.1 mL), using 0.5
mL of either 10~2 or 10~3 dilution per swab, 3,500 or 350 PFU, respectively. TCID50/0.1 mL with SD = 0.71-1.42 x 105 and
PFU/0.1 mL with SD = 4.95-9.90 x 105.
** Values are based on TCID50 analysis of UF-retentates from replicate swabs, where PFU/Swab = TCID50/Swab x 0.7.
Avg. and SD of three PFU/Swab values for each viral dilution tested.
*** Values are based on actual measurement of recovered virus by TCID50 analysis for swabs spiked with 3500 and 350
PFU and calculated for swabs spiked with 175, 35 or 18 PFU using an average recovery efficiency (determined from the
other virus dilutions). Recovered PFU were split between T0 and T9 wells.
NA = Not Applicable; This viral dilution was not used to spike swabs.
Not Determined; Recovery efficiency was not determined directly from these viral dilutions.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Avg. = Average; SD = Standard Deviation.
69

-------
Table 49 RV-RT-PCR Results (N1 Assay) for SARS-CoV-2-Spiked Swabs Processed and
Used to Infect Vero E6 Cells with 2-hr Infection - Replicate Experiment
Kslimnled
Slnrling
PI-1 /
Sw ;t h
Ksliniiiled
A\«. PI !
lor RY-
Ri-P(R
An iilvsis- -
Swsih
Replicnle
R1-PC R
Replicate
C'i hv Post-1 nI'oclion
Incubation Timepoinl
All or 2-hr In I'ecl ion
A\g. AC,
(SI))-""-
I",,
1,
175
107
1
1
29.8
19.1
10.9(0.1)
2
30.1
19.1
3
30.0
19.0
Avg. (SD)
30.0 (0.2)
19.1 (0.03)
2
1
30.3
17.8
12.5 (0.1)
2
30.4
17.8
3
30.2
17.9
Avg. (SD)
30.3 (0.1)
17.8 (0.06)
3
1
29.9
19.1
10.8 (0.1)
2
29.8
19.2
3
29.9
18.9
Avg. (SD)
29.9 (0.04)
19.1 (0.1)
(herall A\«.(SD)
30.0 (0.2)
18.7(0.6)
11.4(0.8)
35
21
1
1
32.9
20.2
12.9 (0.2)
2
33.3
20.1
3
32.9
20.3
Avg. (SD)
33.1 (0.2)
20.2 (0.1)
2
1
33.6
20.1
13.6(0.2)
2
34.1
20.2
3
33.6
20.2
Avg. (SD)
33.8 (0.3)
20.2 (0.04)
3
OM'lilll
1
32.7
19.4
13.5 (0.3)
13.3(0.4)
2
32.7
19.3
3
33.4
19.5
Avg. (SD)
A\g. (SI))
32.9 (0.4)
33.3 (0.5)
19.4 (0.1)
!«>.«> (0.4)
18
11
1
1
32.8
36.3
-2.8 (0.5)
2
33.7
36.0
3
33.9
36.5
Avg. (SD)
33.5 (0.6)
36.3 (0.3)
2
1
32.6
35.0
-2.0 (0.2)
2
33.0
34.9
3
32.8
34.5
Avg. (SD)
32.8 (0.2)
34.8 (0.3)
3
1
33.5
35.8
-4.5 (1.5)
2
33.0
40.0
3
33.8
37.9
Avg. (SD)
33.4 (0.4)
37.9 (2.1)
(herall A\«.(SI»
33.2 (0.5)
36.3 (1.7)
-3.1 (1.4)
* Values are based on dilutions from a SARS-CoV-2 titered stock (TCID50 105 °/0.1 mL or 7.0 x 104 PFU/0.1 mL).
Estimated PFU/Swab = 0.7 x TCID50/Swab.
** Avg. values are based on SARS-CoV-2 recovery determined from replicate swabs (Table 48).
*** Avg. and SD are from triplicate RT-PCR analyses per swab replicate. The Overall Avg. and SD are based
on three replicate swabs.
70

-------
**** sd represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the T9 values squared)/2.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. =
Average; SD = Standard Deviation.
Table 50 RV-RT-PCR Results (N2 Assay) for SARS-CoV-2-Spiked Swabs Processed and
Used to Infect Vero E6 Cells with 2-hr Infection - Replicate Experiment

Ksliniiilcd


C'i hv Posl-lnlection

Ksliiiiiilod
Pi t /
A\j». PI-1
lor RY-
Swsih
Replic:ilo
R1-PC R
Rcpliciilc
1 lieu bill ion
After 2-hr
Timcpoinl
In lection
A\g. AC 'i
(si) )'¦•'¦•'¦•
Sw ;i h
RT-I'CR
An silvsis- -
In
1,



1
28.3
17.3



1
2
28.3
17.2
11.1 (0.1)


3
28.4
17.0



Avg. (SD)
28.3 (0.1)
17.2 (0.1)




1
28.4
15.6



2
2
28.6
15.7
12.8 (0.1)
175
107
3
28.6
15.8



Avg. (SD)
28.5 (0.1)
15.7 (0.07)




1
28.5
17.0



3
2
28.4
16.9
11.5 (0.04)


3
28.5
17.0



A\«. (SI))
28.5 (0.04)
16.') (0.04)



()\it:iII
A\g. (SI))
28.4(0.1)
16.6 (0.7)
11.8(0.8)



1
31.5
18.0



1
2
31.6
17.9
13.7(0.1)


3
31.8
17.9



Avg. (SD)
31.6 (0.2)
17.9 (0.1)




1
32.5
17.8



2
2
31.8
17.8
14.4 (0.3)
35
21
3
32.4
17.8



Avg. (SD)
32.2 (0.4)
17.8 (0.02)




1
31.5
17.3



3
2
31.0
17.0
14.2 (0.2)


3
31.4
17.1



A\«. (SD)
31.3(0.3)
17.1 (0.1)



()\erall
A\g. (SI))
31.7(0.5)
17.6 (0.4)
14.1 (0.4)



1
31.7
34.2



1
2
31.6
35.5
-3.1 (0.5)


3
31.7
34.8



Avg. (SD)
31.7 (0.1)
34.8 (0.7)




1
31.0
33.7



2
2
30.9
33.5
-2.6 (0.2)
18
11
3
31.3
33.7



Avg. (SD)
31.1 (0.2)
33.6 (0.2)




1
31.7
34.4



3
2
31.8
37.0
-3.3 (1.1)


3
32.0
34.1



Avg. (SD)
31.8 (0.1)
35.1 (1.6)



()\it:iII
A\g. (SI))
31.5(0.4)
34.5(1.1)
-3.0 (0.*7)
71

-------
* Values are based on dilutions from a SARS-CoV-2 titered stock (TCID50 105 °/0.1 mL or 7.0 x 104 PFU/0.1 mL).
Estimated PFU/Swab = 0.7 x TCID50/Swab.
** Avg. values are based on SARS-CoV-2 recovery determined from replicate swabs (Table 48).
*** Avg. and SD are from triplicate RT-PCR analyses per swab replicate. The Overall Avg. and SD are based
on three replicate swabs.
**** represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the T9 values squared)/2.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. = Average; SD
= Standard Deviation.
Table 51 Summary of RV-RT-PCR Avg. Ct and ACt Results for SARS-CoV-2-Spiked Swabs
Processed and Used to Infect Vero E6 Cells with 2-hr Infection (N1 Assay) - Replicate Experiment
Ksliimilcd
Slsirling
PI-1 /
Sw ;i h
Ksliniiilcd
A\«. 1*1" 1
lor RY-
RT-IH R
Analysis1
Swsih
Replicate
A\». (SI)) C'i by Post-
Infection Incubation
Timcpoint Alter 2-hr
Inlcction	
A\». A( 'i
(SI)) "
T„
T,
175
107
1
30.0 (0.2)
19.1 (0.03)
10.9(0.1)
2
30.3 (0.1)
17.8 (0.06)
12.5 (0.1)
3
29.9 (0.04)
19.1 (0.1)
10.8 (0.1)
\\». (SI))
30.0 (0.2)
IS.7 (0.6)
11.4 (O.S)
35
21
1
33.1 (U.2)
2U.2 (U.l)
12.9 (0.2)
2
33.8 (0.3)
20.2 (0.04)
13.6(0.2)
3
32.9 (0.4)
19.4 (0.1)
13.5 (0.3)
Ay... (SI))
33.3 (0.5) 19/) (0.4)
13.3(0.4)
18
11
1
33.5 (0.6)
36.3 (0.3)
-2.8 (0.5)
2
32.8 (0.2)
34.8 (0.3)
-2.0 (0.2)
3
33.4 (0.4)
37.9 (2.1)
-4.5 (1.5)
A\g. (SI))
33.2 (0.5)
36.3 (1.7)
-3.1 (1.4)
* Values are based on dilutions from a SARS-Co\ -J" liloivd slock (TCII)^n in n.lmLor"n In Pl'U/0.1 mL).
Estimated PFU/Swab = 0.7 x TCID50/Swab.
** Avg. values are based on SARS-CoV-2 recovery determined from replicate swabs (Table 48).
*** Avg. and SD are from triplicate RT-PCR analyses per swab replicate.
**** represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the T9 values squared)/2.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. =
Average; SD = Standard Deviation.
72

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Table 52 Summary of RV-RT-PCR Avg. Ct and ACt Results for SARS-CoV-2-Spiked Swabs
Processed and Used to Infect Vero E6 Cells with 2-hr Infection (N2 Assay) - Replicate Experiment
KsliniiiU'd
Stjirliii"
PI-1 /
Sw ;i h
Kslimiiled
A\«. 1*11
lor RY-
RT-IH R
Analysis'-
Swjib
Replic;ite
A\». C'i hv I'osl-lnfeclion
1 ncuh;ition l iinopoint Alter
2-hr Infection""ft
A\j>. AC i
(SI) )'¦•'¦'•'¦•¦•
'I'm
1,
175
107
1
28.3 (0.1)
17.2 (0.1)
11.1 (0.1)
2
28.5 (0.1)
15.7(0.07)
12.8 (0.1)
3
28.5 (0.04)
16.9 (0.04)
11.5 (0.04)
A\«.(SI»
28.4 (0.1)
!(>.(> (0.7)
11.8 (0.8)
35
21
1
31.6(0.2)
17.9 (0.1)
13.7 (0.1)
2
32.2 (0.4)
17.8 (0.02)
14.4 (0.3)
3
31.3 (0.3)
17.1 (0.1)
14.2 (0.2)

A\». (SI)) 31.7(0.5) 17.0(0.4)
14.1 (0.4)
18
11
1
31.7(0.1)
34.8 (0.7)
-3.1 (0.5)
2
31.1 (0.2)
33.6 (0.2)
-2.6 (0.2)
3
31.8 (0.1)
35.1 (1.6)
-3.3 (1.1)
Ay... (SI))
31.5(0.4)
34.5(1.1)
-3.0 (0.7)
* Values are based on dilutions from a SARS-CoV-2 titered stock (TCID50 105 °/0.1 mL or 7.0 x 104 PFU/0.1 mL).
Estimated PFU/Swab = 0.7 x TCID50/Swab.
** Avg. values are based on SARS-CoV-2 recovery determined from replicate swabs (Table 48).
*** Avg. and SD are from triplicate RT-PCR analyses per swab replicate.
**** represents the pooled SD, which equals the square root of the following: (SD for T0 values squared plus the SD for
the T9 values squared)/2.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. =
Average; SD = Standard Deviation.
The results for RV-RT-PCR analysis of swabs, like that for swab swatches, showed the optimized RV-
RT-PCR method reproducibly exhibited a less than 50-virion) sensitivity of detection using the CDC-
developed N1 and N2 assays. Considering front-end sample processing of ~3 hr, along with 2 hr viral
infection in Vero E6 cells, 9 hr post-infection incubation, and finally, 3 hr for RNA extraction and RT-
PCR analysis, swab spiked with -35 PFU of SARS-CoV-2 showed detection in a total time-to-results of
just 17 hours (Table 53 and Table 54). It should be noted, however, that this total time does not include
the time needed to prepare the Vero E6 cells and cell culture plates for sample testing. During an
unforeseen/unexpected need for sample analysis for infectious SARS-CoV-2, the cell stock will need to
be prepared, and seeding density in 96-well plates will need to be determined. Furthermore, the cells
will need to be plated 18-24 hr in advance, before the processed sample can be used to infect cell culture
for RV-RT-PCR-based analysis. However, once these conditions are fulfilled (i.e., culturing Vero E6
cells), RV-RT-PCR-based sample analysis can be planned such that continuous batches of samples can
be accommodated for analysis without the time needed for cell culture preparation.
73

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Table 53 Combined RV-RT-PCR Results from Replicate Experiments for
SARS-CoV-2-Spiked Swabs Processed and Used to Infect Vero E6 Cells with
2-hr Infection and 9-hr Post-Infection Incubation - N1 Assay
Ksliiiinloil
St.irtiii" PI-1 /
Swiilv-
Kst i ill ;i led
I'l l /Swjib lor
RY-RT-IH R
Analysis•
A\». AC 'i
(SI) )••••• ••
I'ositiM' Results
620
220
11.0 (0.6)
3 of 3
310
139
10.9 (0.1)
3 of 3
175
107
11.4 (0.5)
3 of 3
62
28
12.7 (0.6)
3 of 3
35
21
13.3 (0.4)
3 of 3
18
11
-3.1 (1.7)
Oof 3
* Values are based on dilutions from a SARS-CoV-2 titered stock.
Estimated PFU/Swab = 0.7 x TCID50/Swab.
** Average values are based on SARS-CoV-2 recovery determined from replicate swabs
(Tables 43 and 48).
*** SD represents the pooled SD, which equals the square root of the following: (SD for T0
values squared plus the SD for the T9 values squared)/2.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. =
Average; SD = Standard Deviation.
Table 54 Combined RV-RT-PCR Results from Replicate Experiments for
SARS-CoV-2-Spiked Swabs Processed and Used to Infect Vero E6 Cells with
2-hr Infection and 9-hr Post-Infection Incubation - N2 Assay
KslimnU'il
Stsirtin*> I'l'l /
Swiilv-
Ksliimilcd A\».
PI-1 lor RY-RT-
l>( R An;tlysis
A\». AC'i
(SI) )••••• ••
I'ositiM' Results
620
220
11.5 (U.t>)
3 of 3
310
139
11.2 (0.2)
3 of 3
175
107
11.8 (0.5)
3 of 3
62
28
13.2 (0.5)
3 of 3
35
21
14.1 (0.4)
3 of 3
18
11
-3.0 (0.7)
Oof 3
* Values are based on dilutions from a SARS-CoV-2 titered stock.
Estimated PFU/Swab = 0.7 x TCID50/Swab.
** Avg. values are based on SARS-CoV-2 recovery determined from replicate swabs
(Tables 43 and 48).
*** SD represents the pooled SD, which equals the square root of the following: (SD for T0
values squared plus the SD for the T9 values squared)/2.
PFU = Plaque Forming Units; TCID50 = 50% Tissue Culture Infectious Dose; Ct = Cycle Threshold; Avg. =
Average; SD = Standard Deviation.
74

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5.0
Summary and Conclusions
At the onset of the COVID-19 pandemic, the EPA Office of Research and Development, Homeland
Security Research Program, expanded their research horizons to respond to this public health
emergency. One of the urgently undertaken research projects was to develop a rapid analytical method
to detect infectious SARS-CoV-2 in environmental samples to support and expedite the studies on
understanding the surface transmission of the virus and to aid environmental epidemiological
investigations. A proof-of-concept RV-RT-PCR method was expeditiously developed. This report
describes the method development research. The RV-RT-PCR method will allow detection of infectious
SARS-CoV-2 from environmental surface samples and similar sample types within hours, rather than
several days required by currently used cell-culture-based methods.
Briefly, the SARS-CoV-2 RV-RT-PCR method integrates cell-culture based enrichment of the virus in a
sample with virus-gene-specific RT-PCR-based molecular analysis. RT-PCR analysis of SARS-CoV-2
RNA is conducted both before and after enrichment of the virus in cell-culture to determine the cycle
threshold (Ct) difference (ACt). An algorithm based on ACt > 6 representing ~ 2-log or more increase
in SARS-CoV-2 RNA following enrichment determines the presence of infectious virus in the sample.
An additional feature that contributes to the rapidity of this method is that the post-enrichment RT-PCR
analysis is performed while the virus is replicating in the host cells and not after complete cell lysis
resulting in detection of the virus in hours rather than days usually taken by current cell-culture based
methods.
To develop this rapid method using such an algorithm, it was important to first establish the method
logistics using the MHV, which is closely related to SARS-CoV-2 in the Betacoronavirus genus and can
be handled under BSL-2 conditions with fewer laboratory restrictions/controls. Accordingly, in the first
part of this project, the RV-RT-PCR method development was performed using MHV and its host cell
line, 17C1-1. The results of the MHV RV-RT-PCR experiments laid the foundation for development of
the SARS-CoV-2 RV-RT-PCR method, specifically as follows: (a) the method can be performed in 96-
well plates instead of cell-culture flasks/plates by establishing optimum host cells seeding density; (b) a
Promega™ RNA extraction kit and Takara Bio RT-PCR kit could be effectively used; (c) the Amicon®
Ultra-15 centrifugal filter device can be effectively used for sample (virus) cleanup/concentration; (d)
method sensitivity of detection achieved with the virus in suspension can be maintained with swab
swatches used as samples; (e) the RV-RT-PCR method can yield analytical results in hours rather than
several days taken by the traditional cell-culture based method. The MHV RV-RT-PCR method
afforded detection of a low number of virions using a 4-hr virus-host cells infection period and 8-hr
post-infection incubation period for a total time-to-results of about 18 hours.
Based on this foundation, the RV-RT-PCR method was developed for SARS-CoV-2 using Vero E6 cells
in a 96-well plate format. With the SARS-CoV-2 suspension as well as swab swatches spiked with the
virus, the method showed a detection sensitivity of <50 virions per sample using a 2-hr virus-host cell
infection period and 9-hr post-infection incubation period for a total time-to-results of about 17 hours.
Using these parameters, the SARS-CoV-2 RV-RT-PCR method performance was finally evaluated by
spiking the swabs and the method sensitivity to detect infectious virus and the time-to-results were
confirmed. The method performed well with both N1 and N2 RT-PCR assays developed by the CDC.
A flow chart and estimated timeline for real-world sample analysis based on the SARS-CoV-2 RV-RT-
PCR method is shown in Figure 8.
75

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Swab Sample
(in tube w/PBS)
Filter 0.45 |jm Filter 0.22 jjm
Sample Receipt & Processing = ~4 hr
# * •
Vortex-mix UF-Coricentrate
Extract RNA
from T0 plate
'\j r'r\
Extract RNA
from Tf plate
'V"\
RNA Extraction, RTPCR, and
Results Reporting = -4 hr
Cell culture infection
& incubation
Incubate
at 37°C for
9-12 hr
RTPCR on T0 and Tf
Report Results
Infect cell culture:
Split filtrate between
Tq and Tf plates
Total time for 22 samples =
~19-22 hr, with subsequent
batches of 22 samples
every 4-8 hr
Figure 8 SARS-CoV-2 RV-RT-PCR Analysis Flow Chart and Estimated Timeline for Swab Samples.
The SARS-CoV-2 RV-RT-PCR method uses a 96-well plate format for the host cell-virus-culture
resulting in a small footprint, which is highly desirable in a BSL-3 laboratory. The small footprint and
reduced waste generation primarily results from (a) significantly fewer multi-well plates compared to
cell-culture flasks/plates required for traditional methods, (b) lower volumes of growth medium and
reagents, and (c) less solid waste from predominant use of micropipette tips for small volumes rather
than large serological pipets required with the use of traditional methods.
Further research work on the SARS-CoV-2 RV-RT-PCR method is ongoing. The work will focus on
evaluating the method performance in the presence of environmental interferents and inactive SARS-
CoV-2 to mimic natural decay on surfaces. Additionally, the method will be evaluated using actual
surface swab samples.
The SARS-CoV-2 RV-RT-PCR method can greatly reduce the time-to-results from days to hours,
support timely understanding of surface transmission of this virus, and support epidemiology
investigations and environmental surveillance in healthcare and non-healthcare facilities, as well as
within a community. This RV-RT-PCR method can assist public health agencies and potentially
enhance capabilities of laboratories, including public health laboratories, involved in sample analysis
and aid decision makers in planning response efforts. The SARS-CoV-2 RV-RT-PCR method
developed using the non-human primate kidney cell line, Vero E6, can also be adapted to various human
cell lines, including pulmonary (Calu3), intestinal (Caco2), hepatic (Huh7), and neuronal (U251) cells
for potential application for efficacy testing of antibody-based vaccines and anti-viral drugs. Further,
since the propagation of virus through traditional cell culture creates a potential selection pressure,
especially as cultures are prolonged, this rapid method may be useful for characterization of SARS-
CoV-2 variants via specific RT-PCR assays or genome sequencing of RNA from positive RV-RT-PCR
samples. Finally, the method can serve as a model for developing methods for other viruses of concern
including both bioterrorism and public health threats.
76

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6.0
References
1.	Scientific Brief: SARS-CoV-2 and Potential Airborne Transmission. CDC, October 05, 2020 (Last
accessed February 12, 2021).
https://www.cdc.gov/coronavirus/2019-ncov/more/scientific-brief-sars-cov-2.html
2.	COVID-19: Frequently Asked Questions. CDC, December 31, 2020 (Last accessed February 12,
2021).
https://www.cdc.gOv/coronavirus/2019-ncov/faq.html#Spread
3.	DeBiasi RL and Delaney M. Symptomatic and asymptomatic viral shedding in pediatric patients
infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). JAMA Pediatrics,
175, 16-18, 2021.
https://doi.org/10.1001/iamapediatrics.202Q.3996
4.	Li W, Su YY, Zhi SS, Huang J, Zhuang CL, et al. Virus shedding dynamics in asymptomatic and
mildly symptomatic patients infected with SARS-CoV-2. Clin Microbiol Infect, 26(11), 1556.ele-
1556.e6, 2020.
https://doi.Org/10.1016/i.cmi.2020.07.008
5.	Arons MM, Hatfield KM, Reddy SC, Kimball A, James A, et al. Presymptomatic SARS-CoV-2
infections and transmission in a skilled nursing facility. N Engl J Med, 382, 2081-2090, 2020.
https://doi.org/10.1056/NEJMoa2008457
6.	Avanzato VA, Matson MJ, Seifert SN, Pryce R, Williamson BN, et al. Case Study: Prolonged
infectious SARS-CoV2 shedding from an asymptomatic immunocompromised individual with
cancer. Cell 183(7), 1901-1912, 2020.
https://doi.Org/10.1016/i.cell.2020.10.049
7.	Wei WE, Li Z, Chiew CJ, Yong SE, Toh MP, Lee VJ. Presymptomatic transmission of SARS-
CoV-2 - Singapore, January 23-March 16, 2020. Morb Mortal Wkly Rep, 69, 411-415, 2020.
http://dx.doi.org/10.15585/mmwr.mm6914el
8.	He X, Lau EHY, Wu P, Deng X, Wang J, et al. Temporal dynamics in viral shedding and
transmissibility of COVID-19. Nat Med, 26, 1491-1493, 2020.
https://doi.org/10.1038/s41591-02Q-1016-z
9.	van Kampen JJA, van de Vijver DAMC, Fraaij PLA, Haagmans BL, Lamers MM, et al. Duration
and key determinants of infectious virus shedding in hospitalized patients with coronavirus
disease-2019 (COVID-19). Nat Commun, 12, 267, 2021.
https://doi.org/10.1038/s41467-020-2Q568-4
10.	Ferretti L, Wymant C, Kendall M, Zhao L, Nurtay A, et al. Quantifying SARS-CoV-2
transmission suggests epidemic control with digital contact tracing. Science, 368(6491):eabb6936,
2020. https://doi.org/10.1126/science.abb6936
11.	Li W, Lin J, Duan X, Huang W, Lu X, Zhou J, Zong Z. Asymptomatic COVID-19 patients can
contaminate their surroundings: an environment sampling study, mSphere, 5 (3) e00442-20, 2020.
https://doi.org/10.1128/mSphere.00442-20
12.	Kampf G, Bruggemann Y, Kaba HEJ, Steinmann J, Pfaender S, et al. Potential sources, modes of
transmission and effectiveness of prevention measures against SARSCoV-2. J Hosp Inf, 106, 678-
77

-------
697, 2020.
https://doi.Org/10.1016/i.ihin.2020.09.022
13.	Santarpia JL, Rivera DN, Herrera VL, Morwitzer MJ, Creager HM, et al. Aerosol and surface
contamination of SARS-CoV-2 observed in quarantine and isolation care. Sci Rep, 10(1): 12732,
2020.
https://www.nature.com/articles/s41598-02Q-69286-3
14.	Chia PY, Coleman KK, Tan YK, Ong SWX, Gum M, et al. Singapore Novel Coronavirus
Outbreak Research Team. Detection of air and surface contamination by SARS-CoV-2 in hospital
rooms of infected patients. Nat Commun, 11, 2800, 2020.
https://doi.org/10.103 8/s41467-020-16670-2
15.	Guo Z-D, Wang Z-Y, Zhang SF, Li X, Li L, et al. Aerosol and surface distribution of severe acute
respiratory syndrome coronavirus 2 in hospital wards, Wuhan, China. Emerg Infect Dis 26, 1583-
1591, 2020.
https://doi.org/10.3201/eid2607.20Q885
16.	Ong SWX, Tan YK, Chia PY, Lee TH, Ng OT, et al. Air, surface environmental, and personal
protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (SARS-
CoV-2) from a symptomatic patient. JAMA, 323, 1610-1612, 2020.
https://doi.org/10.1001/iama.202Q.3227
17.	Wu S, Wang Y, Jin X, Tian J, Liu J, et al. Environmental contamination by SARS-CoV-2 in a
designated hospital for coronavirus disease 2019. Am J Infect Control, 48, 910-914, 2020.
https://doi.Org/10.1016/i.aiic.2020.05.003
18.	Ye G, Lin H, Chen L, Wang S, Zeng Z, et al. Environmental contamination of SARS-CoV-2 in
healthcare premises. J Infect, 81, el-e5, 2020.
https://doi.Org/10.1016/i.iinf.2020.04.034
19.	Luo L, Liu D, Zhang H, Li Z, Zhen R, et al. Air and surface contamination in non-health care
settings among 641 environmental specimens of 39 COVID-19 cases. PLoS Negl Trop Dis 14(10):
e0008570, 2020.
https://doi.org/10.1371/iournal.pntd.000857Q
20.	Harvey AP, Fuhrmeister ER, Cantrell M, Pitol AK, Swarthout JM, et al. Longitudinal monitoring
of SARS-CoV-2 RNA on high-touch surfaces in a community setting. Environ Sci Technol Lett
8(2), 168-175, 2021.
https://doi.org/10.1021/acs.estlett.0cQ0875
21.	Marques M, Domingo JL. Contamination of inert surfaces by SARS-CoV-2: Persistence, stability
and infectivity. A review. EnvRes, 193, 110559, 2021.
https://doi.Org/10.1016/i.envres.2020.l 10559
22.	Bueckert M, Gupta R, Gupta A, Garg M, and Mazumder A. Infectivity of SARS-CoV-2 and other
coronaviruses on dry surfaces: Potential for indirect transmission. Materials, 13, 5211, 2020.
https://doi.org/10.3390/mal3225211
23.	Bedrosian N, Mitchell E, Rohm E, Rothe M, Kelly C, et al. A systematic review of surface
contamination, stability, and disinfection data on SARS-CoV-2 (Through July 10, 2020). Environ
Sci Technol, Nov 23, 2020.
https://dx.doi.org/10.1021/acs.est.0c05651
78

-------
24.	Meyerowitz EA, Richterman A, Gandhi RT, Sax PE. Transmission of SARS-CoV-2: A Review of
viral, host, and environmental factors. Ann Intern Med, 174, 69-79, 2021.
https://dx.doi.org/10.7326/M20-50Q8
25.	Aboubakr HA, Sharafeldin TA, Goyal SM. Stability of SARS-CoV-2 and other coronaviruses in
the environment and on common touch surfaces and the influence of climatic conditions: A
review. Transbound Emerg Dis, 00, 1-17, 2020.
https://doi.org/10. Ill 1/tbed. 13707
26.	Riddell S, Goldie S, Hill A, Eagles D, Drew TW. The effect of temperature on persistence of
SARS-CoV-2 on common surfaces. Virol J, 17, 145, 2020.
https://doi.org/10.1186/sl2985-020-Q1418-7
27.	van Doremalen N, Bushmaker T, Morris DH, Holbrook MG, Gamble A, et al. Aerosol and surface
stability of SARS-CoV-2 as compared with SARS-CoV-1. N Engl J Med, 382(16), 1564-1567,
2020.
https://doi.org/10.1056/NEJMc2004973
28.	Pastorino B, Touret F, Gilles M, de Lamballerie X, Charrel RN. Prolonged infectivity of SARS-
CoV-2 in fomites. Emerg Infect Dis, 26(9), 2256-2257, 2020.
https://doi.org/10.3201/eid2609.201788
29.	Biryukov J, Boydston JA, Dunning RA, Yeager JJ, Wood S, et al. Increasing temperature and
relative humidity accelerates inactivation of SARS-CoV2 on surfaces. mSphere 5(4):e00441-20,
2020.
https://doi.org/10.1128/mSphere.00441-20
30.	Ben-Shmuel A, Brosh-Nissimov T, Glinert I, Bar-David E, Sittner A, et al. Detection and
infectivity potential of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
environmental contamination in isolation units and quarantine facilities. Clin Microbiol Inf, 26,
1658-1662, 2020.
https://doi.Org/10.1016/i.cmi.2020.09.004
31.	Letant SE, Murphy GA, Alfaro TM, Avila JA, Kane SR, et al. Rapid-viability PCR method for
detection of live, virulent Bacillus anthracis in environmental samples. Appl Environ Microbiol.
77(18), 6570-6578, 2011.
https://doi.org/10.1128/AEM.00623-11
32.	Kane SR, Shah SR, Alfaro TM. Development of a rapid viability polymerase chain reaction
method for detection of Yersiniapestis. J Microbiol Methods. 162, 21-27, 2019.
https://doi.Org/10.1016/i.mimet.2019.05.005
33.	Kane SR, Shah SR, Alfaro TM. Rapid viability polymerase chain reaction method for detection of
Francisella tularensis. J Microbiol Methods. 166, 1057382, 2019.
https://doi.Org/10.1016/i.mimet.2019.105738
34.	Reynolds KA, Gerba CP, Abbaszadegan M, Pepper LL. ICC/PCR detection of enteroviruses and
hepatitis A virus in environmental samples. Can J Microbiol. 47(2),153-7, 2001.
https://doi.org/10.1139/W00-134
35.	Gallagher EM, Margolin AB. Development of an integrated cell culture-real-time RT-PCR assay
for detection of reovirus in biosolids. J Virol Methods. 139(2), 195-202, 2007.
https://doi.Org/10.1016/i.iviromet.2006.10.001
36.	Rigotto C, Victoria M, Moresco V, Kolesnikovas CK, Correa AA, et al. Assessment of adenovirus,
hepatitis A virus and rotavirus presence in environmental samples in Florianopolis, South Brazil. J
79

-------
Appl Microbiol. 109(6), 1979-1987, 2010.
https://doi.Org/10.llll/i.1365-2672.2010.04827.x
37.	Zou J, Otter JA, Price JR, Cimpeanu C, Garcia DM, et al. Investigating SARS-CoV-2 surface and
air contamination in an acute healthcare setting during the peak of the COVID-19 pandemic in
London, Clin Infect Dis, ciaa905, 2020.
https://doi.org/10.1093/cid/ciaa905
38.	Dellanno C, Vega Q, Boesenberg D. The antiviral action of common household disinfectants and
antiseptics against murine hepatitis virus, a potential surrogate for SARS coronavirus. Am J Infect
Control, 37(8), 649-652, 2009.
https://doi.Org/10.1016/i.aiic.2009.03.012
39.	Hulkower RL, Casanova LM, Rutala WA, Weber DJ, Sobsey MD. Inactivation of surrogate
coronaviruses on hard surfaces by health care germicides. Am J Infect Control, 39(5), 401-407,
2011.
https://doi.Org/10.1016/i.aiic.2010.08.011
40.	Reed LJ, Muench H. A simple method of estimating fifty percent endpoints. Am J Hyg. 27, 493-
497, 1938.
41.	Leibowitz J, Kaufman G, Liu P. Coronaviruses: Propagation, Quantification, Storage, and
Construction of Recombinant Mouse Hepatitis Virus. Curr. Protoc. Microbiol. 21:15E. 1.1-
15E.1.46, 2011.
https://doi.org/10.1002/978Q471729259.mcl5e01s21
42.	Hierholzer JC, Killington RA. Virus isolation and quantitation, pp. 25-46, In Virology Methods
Manual, Mahy BWJ, Kangro HO (Eds.), Elsevier Ltd., New York, NY, 1996.
https://doi.org/10.1016/B978-012465330-6/500Q3-8
43.	Case JB, Li Y, Elliott R, Lu X, Graepel KW, et al. Murine Hepatitis Virus nspl4 Exoribonuclease
Activity Is Required for Resistance to Innate Immunity. J Virol, 14, 92(l):e01531-17, 2017.
https://dx.doi.org/10.1128/JVI.01531-17
44.	Lu X, Wang L, Sakthivel SK, Whitaker B, Murray J, et al. US CDC Real-Time Reverse
Transcription PCR Panel for Detection of Severe Acute Respiratory Syndrome Coronavirus 2.
Emerg Infect Dis, 26(8), 1654-1665, 2020.
https://dx.doi.org/10.3201/eid2608.201246
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7.0
Appendix A.
Protocol for Rapid Viability Reverse
Transcriptase Polymerase Chain Reaction
(RV-RT-PCR)-Based Detection of SARS-
CoV-2 from Swab Samples
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Laboratory Set-up
•	PPE (personal protection equipment) appropriate for either Biosafety Level (BSL) 2 or 3:
For BSL2: Lab coat, long pants, closed toe/heel shoes, safety glasses, gloves
For BSL3: Tyvek®suit, Powered Air Purifying Respirator (PAPR) hood, booties, and double
latex or nitrile gloves
•	Bleach wipes and/or fresh bleach solution (1 part bleach + 9 parts water), with label
including technician initials and date of preparation.
•	Clean/bleach-disinfected BioSafety Cabinet (BSC) and bench surfaces.
Note: All sample manipulations except possibly automated nucleic acid
extraction/purification and RT-PCR analysis are performed in the BSC.
General Laboratory Supplies
•	Gloves (e.g., latex, vinyl, or nitrile)
•	Bleach wipes (Dispatch® Cat. No. 69150 or equivalent)
•	Ziploc®bags (large -20" x 28", medium -12" x 16", small -7" x 8")
•	Sharps waste container
•	Absorbent pad
•	Medium and large biohazard bag(s) and twist tie(s) or rubber band(s)
•	Squeeze bottle with 70% isopropyl alcohol
•	Squeeze bottle with deionized (DI) water
•	Autoclave tape
•	Biohazardous autoclave bags
•	Large photo-tray or similar tray for transport
•	Laboratory markers
•	Timer
•	Sterile disposable aerosol filter pipet tips: 1000 |iL, 200 |iL, 100 |iL, and 10 |iL (Rainin, Cat.
No. SR-L1000F, SR-L200F, SR-L100F, GP-10F or equivalent)
•	1.5 mL Eppendorf Snap-Cap Microcentrifuge Biopur® Safe-Lock™ tubes (Fisher Scientific,
Cat. No. 05-402-24B or equivalent)
•	Sterile 50 mL conical tubes (VWR, Cat. No. 21008-951 or equivalent)
•	Sterile 15 mL conical tubes (VWR, Cat. No. 21008-918 or equivalent)
•	Tubes, sterile 2 mL DNase, RNase-free, gasketed, screw caps (National Scientific, Cat. No.
BC20NA-PS or equivalent)
•	10% Bleach (prepared daily) - Prepare bleach solution by adding 1 part bleach and 9 parts
reagent-grade water.
•	Disposable nylon forceps, sterile (VWR Cat. No. 12576-933 or equivalent)
•	50 mL conical tubes, sterile (VWR Cat. No. 21008-951 or equivalent)
•	15 mL conical tubes, sterile (Invitrogen Cat. No. AM12500 or equivalent)
•	Disposable serological pipets, sterile: 50 mL, 25 mL, 10 mL, 5 mL
•	Single 50 mL conical tube holder (Bel-Art Cat. No. 187950001 or equivalent)
•	Screw cap tubes, 2 mL, sterile (VWR Cat. No. 89004-298 or equivalent)
•	96-well tube rack(s) for 2 mL tubes (8 x 12 layout) (Bel-Art Cat. No. 188450031 or
equivalent)
•	2 mL Eppendorf tubes, sterile (Fisher Scientific Cat. No. 05-402-24C or equivalent)
•	96-well 2 mL tube rack (8 x 12 format) (Bel-Art Cat. No. 188450031 or equivalent)
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•	24-well deep well plates, sterile (Millipore Cat. No. AXYPDW10ML24CS or equivalent) -
for suspension cultures
•	Millex-HV Syringe Filter Unit, 0.45 |im, PVDF, 33 mm, gamma-sterilizable, sterilized
(Millipore® Cat. No. SLHVM33RS or equivalent)
•	10-mL Syringe with BD Luer-Lok™ Tip, sterile (VWR Cat. No. 75846-756 or equivalent)
•	Parafilm, clear, 5.1 cm x 76.2 m (2" x 250') (VWR Cat. No. 52858-076 or equivalent)
•	Ultrafree®-CL Centrifugal Filter 0.45 |im pore size, hydrophilic PVDF, 0.5 mL volume, non-
sterile (Millipore® Cat. No. UFC40LH25 or equivalent)
•	Ultrafree®-MC Centrifugal Filter 0.22 |im pore size, hydrophilic PVDF, 0.5 mL volume,
sterile (Millipore® Cat. No. UFC30GV0S or equivalent)
•	Amicon® Ultra-15 Centrifugal Filter Unit, Ultracel-10 regenerated cellulose membrane, 15
mL (Millipore® Cat. No. UFC901096 or equivalent)
•	EnviroMax Sterile Norovirus Detection Swab & Tube (Puritan Cat. No. P25-88060-PF-UW-
DRY or equivalent).
o Sanigen MicroSwab (South Korea) may also be used, as this was suggested by
CDC colleagues as an appropriate substitute.
Supplies for Real-time RT-PCR
•	96-well PCR plates (ABI Cat. No. 4346906 or equivalent)
•	96-well plate holders, Costar®, black (VWR Cat. No. 29442-922 or equivalent)
•	Edge seals for 96-well PCR plates (Adhesive Plate Sealers, Edge Bio Cat. No. 48461 or
equivalent)
•	Foil seals for 96-well PCR plates (Polar Seal Foil Sealing Tape, E&K Scientific Cat. No.
T592100 or equivalent) - for longer storage of the plates
•	Optical seals (ABI Cat. No. 4311971 or equivalent)
•	Plate paddle (VWR Cat. No. 60941-128 or equivalent) for sealing PCR plates with adhesive
seal
•	PCR-grade water
Supplies for Cell Culture
•	Dulbecco's Modified Eagle's Medium (DMEM), 500 mL (Thermo Scientific Cat. No.
10566024 or equivalent)
•	Fetal Bovine Serum (FBS) (VWR Cat. No. 10018-830 or equivalent)
•	Phosphate Buffered Saline (PBS) IX, Biotechnology Grade, 500 mL (VWR Cat. No. 97062-
818 or equivalent)
•	Penicillin-Streptomycin-FNGZ Mix 100 mL (VWR Cat. No. 12001-712 or equivalent)
•	Cell Culture Flask T25 FC TC TRTD 192 FK, sterile (VWR Cat. No. 75874-324 or
equivalent)
•	Cell Culture Flask T-75 FC TC TRTD 80 FK, sterile (VWR Cat. No. 75874-332 or
equivalent)
•	96 -well Cell Culture Plates, sterile (VWR Cat. No. 10062-900 or equivalent)
•	Trypsin EDTA Solution (VWR Cat. No. MSPP-30-2101 or equivalent)
•	Plate Deep Well 2 mL Ind. Wrap Cs. 30 (VWR Cat. No. 47749-930 or equivalent)
•	Reservoir 25 mL Sterile CS200 (VWR Cat. No. 89108-002 or equivalent)
•	Racks for 50 mL centrifuge tubes
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•	Sterile 50 mL conical tubes (VWR Cat. No. 21008-951 or equivalent)
•	Sterile Pipet tips with aerosol filter for 1000 |iL and 100 |iL (Rainin Cat. No. SR-L1000F and
GP-100F or equivalent)
•	Biotransport carrier (Nalgene, Thermo Scientific Cat. No. 15-251-2 or equivalent)
Equipment
•	Biological Safety Cabinet (BSC) - Class II or Class III
•	PCR preparation hood (optional)
•	Shaker incubator for RV-RT-PCR with suspension culture (PlateShake, Perkin Elmer Cat.
No. 1296-003 or equivalent)
•	Analytical balance, with Class S reference weights, capable of weighing 20 g ± 0.001 g
•	Applied Biosystems™ 7500 Fast Real-Time PCR System (Applied Biosystems Inc., Foster
City, CA)
•	Refrigerated centrifuge (Eppendorf Cat. No. 022627023 or equivalent) with PCR plate
adapter (Eppendorf Cat. No. 022638041 or equivalent) and corresponding safety cups or
PCR plate spinner (placed in BSC) (VWR, Cat. No. 89184-608 or equivalent)
•	Refrigerated centrifuge with Rotor A-4-62, 50-mL tube adapter and corresponding safety
cups/lids (Eppendorf Cat. No. 022627023 or equivalent)
•	Microcentrifuge (Eppendorf Model 5415D or equivalent) with 1.5 mL tube rotor
•	Refrigerated micro-centrifuge and rotor for 1.5-2.0 mL tubes (Eppendorf Model 5417R) with
aerosol-tight lid (VWR, Cat. No. 97058-912 or equivalent)
•	Mini plate spinner for PCR plates (Millipore, Labnet MPS 1000, Cat. No. Z723649, or
equivalent)
•	Single-tube vortexer
•	Single-channel micropipettors (1000 \\L, 200 |j,L, 100 |iL, 20 |iL, 10 |iL)
•	Serological pipet aid
•	Incubator(s), microbiological and cell culture (CO2) types, maintained at 37°C
•	Autoclave or steam sterilizer, capable of achieving 121°C (15 psi)
•	Cold block for 1.5 or 2 mL tubes (Eppendorf Cat. No. 3880 001.018 or equivalent)
•	MagNA Pure Compact Automated DNA/RNA Extraction Platform (Roche Applied Sciences,
Indianapolis, IN; Cat No. 03731146001) or other automated system (optional)
Reagents
•	PCR-grade, nuclease-free water, sterile (ThermoFisher Cat. No. 4387936 or equivalent)
•	PBS IX, Biotechnology Grade, 500 mL (VWR Cat. No. 97062-818 or equivalent)
•	TE buffer (IX Tris-HCl-EDTA [Ethylenediaminetetraacetic acid]) buffer, pH 8.0, Fisher
Scientific, Cat. No. BP2473-500 or equivalent)
•	Promega™ MagneSil® Total RNA mini-Isolation System (Promega Cat. No. Z3351 or
equivalent)
•	RNasin® Plus RNase Inhibitor (Promega Cat. No. N2611 [2,500 units], N2615 [10,000
units], or equivalent)
•	100% Ethanol (200-proof) for preparation of 90% ethanol by dilution with PCR-grade water
•	2-propanol for clean-up and RNA isolation procedure
•	MagNA Pure LC Total Nucleic Acid Isolation Kit Lysis/Binding Buffer-Refill (Roche
Applied Sciences, Indianapolis, IN; Cat. No. 03 246 779 001 or equivalent) - optional
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•	MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche Applied Sciences, Indianapolis,
IN; Cat. No. 03730964001 or equivalent) - optional
•	RT-PCR Primers and probe for SARS-CoV-2, N1 and N2 Assays (Biosearch Technologies,
Cat. No. KIT-NCOV-PP1-1000)
•	Primers and probe for SARS-CoV-2, N1 Assay
•	Forward Primer (2019-nCoV_Nl-F) -
5' - GAC CCC AAA ATC AGC GAA AT -3'
•	Reverse Primer (2019-nCoV_N 1 -R) -
5' - TCT GGT TAC TGC CAG TTG AAT CTG -3'
•	Probe (2019-nCoV_Nl-P)-
5'-FAM- ACC CCG CAT TAC GTT TGG TGG ACC -BHQ1-3'
•	Primers and probe for SARVS-CoV-2, N2 Assay
•	Forward Primer (2019-nCoV_N2-F) -
5' - TTA CAA AC A TTG GCC GCA AA -3'
•	Reverse Primer (2019-nCoV_N2-R) -
5' - GCG CGA CAT TCC GAA GAA -3'
•	Probe (2019-nCoV_N2-P) -
5'-FAM- AC A ATT TGC CCC CAG CGC TTC AG -BHQ1-3'
•	Quantitative Synthetic RNA from SARS-Related Coronavirus 2 (BEI Resources, Cat. No.
NR-52358 or equivalent)
Cell Line
• Vero E6 cell line (African green monkey kidney cells; ATCC® CRL-1586™, ATCC;
Manassas, VA)
Cell Culture Preparation and Swab Sample Processing for RV-RT-PCR Analysis
1. Vero E6 cell culture preparation for swab samples:
a.	Thaw a tube of Vero E6 cells stock culture in 37°C water bath.
b.	Add the 1 mL cell suspension into T25 flask with filtered cap with 5 mL
Dulbecco's Modified Eagle's Medium (DMEM) with 10% FBS, incubate
overnight at 37°C with 5% CO2 in a humidified incubator.
c.	If monolayer is more than 90% confluent, remove media, wash with 5 mL PBS,
add 3 mL trypsin, incubate for less than 5 minutes at 37°C in 5% CO2 in a
humidified incubator or until cells dislodge from the flask surface.
d.	Inactivate trypsin by adding 5 mL DMEM with 10% FBS and gently pipet up and
down to break apart clusters of cells. Transfer to a sterile 15 mL conical tube and
centrifuge at 4,000 rpm (-3,200 x g) for 10 minutes at 10°C (Eppendorf 5810R).
e.	Discard supernatant and resuspend cell pellet in 5-10 mL of DMEM with 10%
FBS, transfer to T75 flask and bring the volume up to 25 mL by adding 10% FBS
DMEM, incubate overnight at 37°C in 5% CO2 in a humidified incubator.
f.	Prepare sample 96-well plate layout(s) in MS Excel per the number of samples to
be analyzed.
g.	If monolayer in T75 is at least 90% confluent, trypsinize the cells as above and
suspend in 10 mL DMEM with 10% FBS. Count the cells and dilute to 35,000
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cells per 0.2 mL using DMEM with 10% FBS based on number of wells needed
per sample plate layouts.
h.	Pipet 0.2 mL to each well in 96-well cell culture plates (VWR Cat. No. 10062-
900 or equivalent). Incubate overnight at 37°C in 5% CO2 in a humidified
incubator.
i.	When the monolayer is at least 90% confluent, the cells are infected with
recovered viral UF-retentate from swab (i.e., 0.22-micron filtrate after UF-
concentration) for RV-RT-PCR analysis.
2. Swab Sample Processing
Note: all work with SARS-CoV-2 will be performed under BSL-3 conditions.
For swab sample in tube with swab handle attached to lid (Puritan Cat. No. P25-
88060-PF-UW-DRY or equivalent):
a.	Vortex tubes with swabs for 30 sec. at -3,000-3,500 rpm in 15 sec bursts with 1-2
sec between for 1 min at room temperature.
b.	Optional filtration for samples containing particulates - For each swab sample,
remove the plunger from a 10-mL syringe and place in the syringe wrapper to
keep sterile while filtering the sample. Aseptically attach a 0.45-micron filter disk
to the 10-mL syringe with Luer-Lok™ tip. Place the syringe with filter disk on top
of a sterile 50-mL conical tube with cap removed. Use Parafilm® sealing film to
secure the syringe/filter disk to the top of the tube.
c.	Using a 10-mL sterile serological pipette, transfer the entire sample suspension to
the 10-mL syringe with filter disk attached. Carefully insert the plunger and push
the liquid through the filter into the tube. After filtration, remove the Parafilm®
sealing film and syringe with filter.
d.	Using a 10-mL sterile serological pipette, transfer the entire filtered suspension to
a labeled Amicon® Ultra-15 Centrifugal Filter Unit. Centrifuge at 4,000 rpm
(-3,200 x g) using a Eppendorf 5810R table-top centrifuge at 10°C. Centrifuge
20-30 min to recover retentate and bring volume to -0.2 mL with PBS (± 0.02
mL). Check volume and centrifuge longer if needed to bring volume to -0.2 mL
for To and T9 cell-culture wells in separate 96-well cell-culture plates.
e.	Optional - for samples with higher levels of potential interferents, a wash step
with 5 mL PBS may be included, followed by centrifugation at 4,000 rpm (-3,200
x g) for 10-15 min and 10°C, dependent on the desired retentate volume (-0.2
mL).
f.	Recover retentate in a sterile 1.5-mL microfuge tube and determine volume. If
needed, bring the volume to -0.2 mL with sterile PBS for To and T9 cell-culture
wells in separate 96-well cell-culture plates.
g.	Transfer retentate to a labeled sterile Ultrafree®-MC Centrifugal 0.22 |im filter
insert in tube (Millipore® Cat. No. UFC30GV0S) and cap the tube. Repeat for
each sample. Place tubes into an aerosol-tight rotor and lid in a refrigerated
centrifuge and centrifuge at 7,000 rpm for 1 min at 4°C.
h.	After filtration, remove filter insert with a sterile forceps, cap tube and place on
wet ice, cold block, or in cold rotor. Note: this filtration step is required prior to
adding the sample retentate to cell culture since the UF device is not sterile.
i.	When ready to add sample filtrate, remove To and T9 96-well cell culture plates
from incubator. Carefully aspirate the medium from each well without disturbing
the cell monolayer (do not allow cells to dry).
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j. Pipet ~0.1 mL sample filtrate (half of the total sample filtrate volume) to the
appropriate To and T9 cell culture wells in separate 96-well cell culture plates for
viral infection (according to sample plate layout). Then, add 0.1 mL DMEM with
2% FBS to each well. Incubate plates at 37°C with 5% CO2 in a humidified
incubator for 2 hr.
k. After the 2-hr infection period, carefully aspirate the medium from each well
without disturbing the cell monolayer (do not allow cells to dry).
1.	First, perform a wash step by adding 0.1 mL 2% FBS DMEM to each
well (for To and T9 plates), and carefully remove this medium. Then,
add 0.1 mL 2% FBS DMEM to each well.
2.	For the To plate(s), carefully remove the medium and add 0.1 mL PBS to
each well. Store plate(s) at -80°C until RNA extraction is conducted.
3.	For the T9 plate(s), incubate for 9 hr at 37°C with 5% CO2 in a
humidified incubator.
4.	After the 9-hr incubation, carefully remove the medium and add 0.1 mL
PBS to each well. Store plate(s) at -80°C until RNA extraction is
conducted.
RNA Extraction
Process To and T9 plates (post-infection incubation plates) for RNA extraction using the
Promega MagneSil® Total RNA mini-Isolation System Kit following manufacturer's
protocol, adapted for 1.5- or 2.0-mL tubes.
a.	Prepare fresh DNase I Solution (for later use, following kit instructions) for the
number of samples to be extracted. (Note: the DNase I solution, when combined
with MnCh and Yellow Core Buffer, cannot be stored).
Combine the following in order for 96 samples (or per sample in parentheses):
•	5.2 mL Yellow Core Buffer (use 5.2 mL/96 or 54.17 [xL/sample)
•	575 [iL MnCh (use 575/96 or 6 [xL/sample)
•	275 [iL DNase I (Prepare DNase I by adding 275 [xL RNase free water to
the bottle containing lyophilized DNase I, and use 275/96 or 2.86
[xL/sample).
b.	Prepare DNase Stop Solution (for later use, following kit instructions). Add 40
mL of 90% ethanol to the DNase Stop Solution bottle and mark when added.
This solution is stable at 22-25°C when tightly capped.
c.	Thoroughly resuspend the MagneSil® RNA Paramagnetic Particles (PMPs) in the
reagent bottle.
d.	Dispense 30 [xL of PMPs to each empty tube (1.5 or 2.0 mL RNase-free tube)
labeled with sample ID.
e.	Place on magnet stand and remove PMP storage buffer.
f.	Cell Culture/Virus Lysis and Capture of Nucleic Acids. Add 0.1 mL RNA Lysis
Buffer to each sample well (To and T9 cell culture wells for each sample) and mix
by pipetting up and down 4 or more times. Transfer well contents to tube with
PMPs. Vortex at -3,000-3,500 rpm using 15 sec bursts with 5-10 sec in between
for 1 minute.
g.	Place on magnet for 1 minute. Remove and discard the supernatant.
h.	Wash. Add 0.1 mL of 90% ethanol to each tube. Vortex at -3,000-3,500 rpm
using 15 sec bursts with 5-10 sec in between for 1 minute.
i.	Place on magnet for 1 minute. Remove and discard the supernatant. Leave to dry
in air for 1 minute or until beads appear dry.

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j. DNase Treatment. Add 50 [xL of prepared DNase I Solution to each tube.
Incubate for 10-15 minutes at room temperature, with intermittent vortexing for
10-15 sec. at -3,000-3,500 rpm every 2-3 min.
k. DNase Inactivation. Add 0.1 mL DNase Stop Solution to each tube and vortex at
-3,000-3,500 rpm using 15 sec bursts with 5-10 sec in between for 2 minutes.
1. Place on magnet for 1 minute, discard the supernatant.
m. Wash. Add 0.1 mL of 90% ethanol to each tube. Vortex for 1 minute at -3,000-
3,500 rpm using 15 sec bursts with 5-10 sec in between. Place on magnet for 1-
minute discard the supernatant,
n. Repeat wash. Leave to dry in air for 2 minutes, or until beads appear dry.
o. RNA Elution. Prepare sufficient Nuclease-Free Water containing RNasin Plus
RNase Inhibitor. Use 0.5 [xL RNasin Plus RNase Inhibitor (Cat. No. N2611,
2,500 units) or 0.125 [xL RNasin Plus RNase Inhibitor (Cat. No. N2615, 10,000
units) per 50 [xL for each tube. Vortex for 2 minutes at -3,000-3,500 rpm using
15 sec bursts with 5-10 sec in between,
p. Place on magnet for 1 minute. Remove supernatant to a new labeled tube,
q. Store the purified total RNA at -80°C, if not ready to perform RT-PCR analysis
immediately. Keep RNA extracts on ice while preparing RT-PCR reagents.
Optional: Automated DNA/RNA Extraction/Purification Procedure - Roche MagNA Pure
Compact
1.	Adapt the Reagent Cartridges (MagNA Pure Compact Nucleic Acid Isolation Kit I; Cat. No.
03730964001) to room temperature (+15 to +25°C) before use. If you use the reagents at
temperatures outside the recommended range, the kit may not work well.
2.	To perform total nucleic acid isolation from mammalian serum/plasma samples with the MagNA
Pure Compact Nucleic Acid Isolation Kit I, different pre-installed purification protocols are
available. The kit is designed for 32 isolations (4 x 8) from 0.1 mL suspension of cultured cells
(containing no more than 1 x 106 cells in PBS). For each protocol, sample and elution volumes
must be chosen from the software menu.
3.	Using these protocols, samples are lysed manually, outside the MagNA Pure Compact
Instrument in a BSC (for BSL3 conditions). Lysates are then transferred to the instrument and
purification is carried out automatically (after a viral inactivation procedure has been validated).
This procedure allows to physically separate the lysis step from the purification step and to load
inactivated sample material to the MagNA Pure Compact Instrument (e.g., when using
potentially infectious sample material). The MagNA Pure LC Total Nucleic Acid Isolation Kit
Lysis/Binding Buffer-Refill (Cat. No. 03246779001) will be used with the
Total NA Plasma external lysis purification protocol for the initial lysis step as described
below.
Optional - MagNA Pure Compact Protocol Using the MagNA Pure Compact Nucleic Acid Isolation Kit
I with LC Total Nucleic Acid Isolation Kit Lysis/Binding Buffer and the Total NA
Plasma external lysis purification protocol
Note: it may be necessary to centrifuge the cells to remove culture medium, then resuspend the cell
pellet in 0.1 mL PBS.
1. Thaw To and T9 plates, if they were stored at -20°C.
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2.	Add 0.1 mL MagNA Pure LC Total Nucleic Acid Isolation Kit Lysis/Binding Buffer-Refill (Cat.
No. 03 246 779 001 or equivalent) to each sample well (To and T9 wells) and mix thoroughly by
pipetting up and down 4 or more times. Transfer well contents to 2-mL screw-cap tube.
3.	Add additional 0.2 mL Lysis/Binding Buffer to each screw-cap tube.
4.	Vortex 10 sec (-3,000-3,500 rpm) to mix.
5.	Incubate tubes for 30 min at room temperature. Every 5 min invert tubes several times to mix.
Alternatively, a rolling tube incubator can be used for continuous mixing. This step ensures
complete inactivation of samples.
6.	Turn on the MagNA Pure Compact instrument.
7.	Remove Cartridge Rack and Tube Rack (with Elution Tube Rack) from the instrument.
8.	Click the Run button on the Main Menu Screen to access Sample Ordering Screen 1.
9.	Follow the software-guided workflow.
10.	Remove a prefilled Reagent Cartridge from the blister pack.
11.	Check the cartridge integrity and filling volumes of the wells. Do not use cartridges that have a
different pattern of filling or that are damaged.
12.	Adapt the Reagent Cartridge to room temperature (30 minutes).
13.	Handle each Reagent Cartridge prior to use as follows:
14.	Always wear gloves when handling the MagNA Pure Compact Cartridge.
a.	Hold the cartridge only at the barcode imprinted area and the opposite side.
b.	Avoid touching the sealing foil covering the cartridge wells.
c.	Avoid touching the two single open wells and do not use them as handles.
d.	Avoid any foam formation and let the fluid within the cartridge wells settle again
completely. If fluid remains under the sealing foil, knock the cartridge bottom gently on
a flat lab bench surface. This is especially important for well 1, which contains a small
volume of Proteinase K.
15.	Scan the barcode.
16.	With the two isolated wells pointing away from you, insert all the wells on the Reagent Cartridge
into the holes in the Cartridge Rack.
17.	Use the guide slots on the rack to help position the cartridge.
18.	Repeat the steps above for the desired numbers of samples (1 to 8).
19.	Proceed to Sample Ordering Screen 2.
20.	Select the appropriate purification protocol from the Protocol menu (Total NA_
Plasmaexternallysis purification protocol).
21.	Select the sample volume (500 [xL).
22.	Select the elution volume (100 |iL),
23.	Select the Internal Control (IC) Volume (0 (iL).
24.	Note: The program allows for inclusion of an IC although it is not requiredfor the proposed
effort.
25.	Insert the appropriate number of Tip Trays (one per purification) into the assigned position in the
instrument Tip Rack.
26.	Check if the tip tray holds a disposable in each position (tip or piercing tool). Do not use tip
trays that are not assembled accordingly.
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27.	Handle Tip Trays with care to prevent tips or piercing tool from falling out of the tray. Should
this happen, discard the respective tip tray and tips. Use the Tip Tray Kit to replace missing Tip
Trays.
28.	Proceed to Sample Ordering Screen 3.
29.	Scan the sample barcode from the primary sample tube or enter the sample name.
30.	Arrange the Sample Tubes in row 1 of the Tube Rack. Make sure the brim of the tubes seats
solidly on the rack.
31.	Pipet the samples into their respective Sample Tubes.
32.	Proceed to Sample Ordering Screen 4.
33.	This screen only appears and accepts information about IC Tubes if you selected a protocol with
internal controls on Sample Ordering Screen 2. The program will skip this screen and proceed
directly to Sample Ordering Screen 5 if you selected a protocol with no IC.
34.	Identify the tube by attaching your own barcode label or writing an ID number on the tube with a
permanent marker.
35.	Put the filled control tubes in row 2 of the Tube Rack.
36.	Insert the Tube Rack with samples.
37.	Reinsert the Tube Rack into the instrument.
38.	Proceed to Sample Ordering Screen 5.
39.	Scan the bar codes of the Elution Tubes.
40.	Place the Elution Tubes into the Elution Tube Rack. Make sure the brim of the tubes seats
solidly on the rack.
41.	Reinsert the Elution Tube Rack into the instrument.
42.	On the Confirmation Screen, check the information display.
43.	If the information is correct, confirm it by touching the "Confirm Data" button, close the front
cover, and start the run.
44.	After the purification run has ended, the Result Screen appears showing the result of the isolation
process for each channel:
45.	The result will be PASS if the isolation run was completed without any warning or error.
46.	The result will be FAIL if any interruption of the process or error occurred during the run. For
each FAIL result, the result screen will show a brief error or warning messages to help you
decide whether the error or warning can be ignored. Refer to the troubleshooting section of the
MagNA Pure Compact Operator's Manual.
47.	Close the Elution Tubes with the supplied tube caps and remove the Elution Tube Rack or the
Elution Tubes immediately after the end of the purification run.
48.	If not proceeding directly to your downstream application, store RNA eluates at -80°C.
49.	Optionally: Start the automated liquid waste discard. Disregard this step if the automated liquid
waste discard is not used.
50.	Empty the MagNA Pure Compact Waste Tank after every purification run if the automated
liquid waste discard is used. Treat liquid waste as potentially infectious (depending on sample
material), and hazardous, since lysis buffers are present.
51.	Disinfect liquid waste by reacting with sufficient freshly-prepared bleach to yield a final
concentration of 10% for 30 min.
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52.	Disinfect external and internal surfaces of the instrument with freshly prepared 10% bleach (with
30 min contact time), followed by isopropanol or water rinse to remove bleach residual.
53.	Autoclave all solid waste in a permitted autoclave according to the Select Agent Facility
requirements.
Note: Store RNA extracts at -80°C until they are used for RT-PCR analysis.
Cleanup Procedure
•	Dispose of all biological materials in autoclave bags (double bagged) and sealed.
•	Autoclave all waste materials.
•	Decontaminate counters and all equipment with bleach (1 volume water and 9 volumes
commercial bleach) made fresh daily. Follow this with rinsing by 70% isopropanol and/or by
rinsing with deionized water.
Real-time RT-PCR Analysis
Note: An RT-PCR Kit will be selected in this effort. RT-PCR Master Mix is typically 2Xor 4Xin
concentration.
Preparation ofprimers and probes from lyophilized stocks:
1.	Locate information from manufacturer's packing slip and document the following information
for preparation of reconstituted primer/probe stocks from lyophilized reagents: date, preparer,
manufacturer name, catalog no., sequence name, scale of synthesis, order date, nmol of
lyophilized material).
2.	Add TE buffer to tube to obtain desired final concentration of primer or probe (i.e., 20 |jM for
primers and 5 |jM for probes).
Note: the reconstitution volume was 0.5 mL for the Biosearch Technologies KlT-nCOV-PPI-
1000for N1 and N2 assay reagents.
3.	Let tube sit at room temperature for 5 min (in the dark since probe is light-sensitive). Mix using
low-moderate vortex (-1,000 rpm).
4.	Invert tube after vortexing (to resuspend any material in the tube cap). Briefly centrifuge (-100
rpm for 3-5 sec) to bring liquid to bottom of the tube.
5.	Prepare aliquots of the 20 |iM primer/0.5 |iM probe stock (at appropriate volumes), and store
aliquot tubes at -20°C (to minimize freeze-thaw cycles). For example, for 100 reactions, -65 [xL
is required.
6.	Since the probe is light-sensitive, store reconstituted primer/probe aliquots in light-protected
container at -20°C.
Preparation of RT-PCR Master Mix and RT-PCR Analysis
1.	Prepare RT-PCR Mix according to the table below (One Step RT-PCR Mix for SARS-CoV-2 N1
and N2 Assays).
2.	Set up 96-well PCR plate with RT-PCR mix according to plate layout in PCR-preparation hood,
seal, and transfer to BSC. Note that RT-PCR analysis may need to be conducted under BSL-3
conditions, depending on biosafety requirements.
3.	Analyze To and T9 RNA extracts for each sample in triplicate and on the same PCR plate,
following the RT-PCR plate layout.
4.	If sample RNA extracts were frozen, transfer them to the biosafety cabinet and let them thaw on
ice or cold block.
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5.	Briefly vortex each RNA extract tube at -1,000-1,500 rpm for 5 sec. and spin tube in small
microcentrifuge (in BSC) briefly (3-5 sec), if needed to ensure the RNA extract is collected at
the bottom of the tube and liquid is not in the lid or on the tube sides.
6.	After adding RNA extracts to the appropriate PCR plate wells, seal the plate with an optical seal
making sure to completely cover all well edges. Use plastic plate paddle or adhesive film
applicator, as appropriate.
7.	Centrifuge sealed plate using mini plate spinner at 500 x g (2,500 rpm) for 1 min.
8.	Run RT-PCR cycle (see below for RT-PCR Thermal Cycling Conditions).
9.	After PCR cycles completion, discard sealed PCR plate to waste. Autoclave. PCR plates with
amplified product are never to be opened in the laboratory.
10.	Follow laboratory cleanup procedure.
One Step RT-PCR Master Mix for SARS-CoV-2 N1 and N2 Assays
Uoitiicnl
Volume (ill.)
1- iiiiil CoiHTnlnilion (11M)
2X PrimeScript III (Takara Bio)
12.5
IX
Primers (20 (j,M) and Probe (5 |iM) Stock
0.625
0.5 (primers)
0.125 (probe)
Molecular Biology Grade Water
6.375
N/A
ROX II (5 OX)
0.5
IX
Template RNA
5
Variable
TOTAL
25

RT-PCR Thermal Cycling Conditions - N1 and N2 Assays for SARS-CoV-2
Slops
RT 1 iicu hilt ion
T;iq Aclix :ilion
l>( R. 45 cvcles
Mold
Mold
l)cii;iliir;ilion
Aiinc;ilin<>/
Kxlcnsion
Temperature
50°C
95°C
95°C
55°C
Time
10 min
2 min
3 sec
30 sec
Standard Ramp Rate.
RV-RT-PCR Data Interpretation
RV-RT-PCR algorithm for a positive virus detection result: An average Ct is determined from
triplicate PCR reactions for To (Time 0 for post-infection incubation) and T9 (9-hr post-infection
incubation) RNA extracts of each sample. The average Ct of the T9 RNA extract is subtracted from the
average Ct of the To RNA extract to determine the average ACt for the sample. The pooled SD for the
average ACt is calculated as the square root of the following: [SD for Ct(To) values squared plus the SD
for the Ct(T9) values squared]/2, where T9 is the 9-hr post-infection incubation period. An algorithm
based on an approximately 2-log or more increase in viral RNA following enrichment is applied such
that the resultant ACt> 6 determined the presence of infectious virus in the sample.
If there is no CTfor the To or T9 RNA extracts (i.e., the result is non-detect), the Ct is set to 45 (the total
number of PCR cycles used) in order to calculate the ACt value. A minimum of two out of three To
PCR replicates with Ct values < 44 (in a 45-cycle PCR) is required to calculate the average Ct. In
addition, a minimum of two out of three T9 PCR replicates is required to calculate the average Ct. Also,
for the sample RT-PCR data to be valid, (1) the negative controls should not yield any measurable Ct
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values (i.e., a minimum of 2 of 3 RT-PCR replicates needed to be non-detect), and (2) no-template RT-
PCR controls should not yield measurable Ct values.
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vvEPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300

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