EPA/600/R-16/313 | September 2016
www.epa.gov/homeland-security-research
United States
Environmental Protection
Agency
oEPA
Development and Optimization
of a Rapid Viability
Polymerase Chain Reaction
(RV-PCR) Protocol for
Detection of Yersi in
Water Samples
Office of Research and Development
Homeland Security Research Program
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EPA/600/R-16/313
September 2016
Development and Optimization of a Rapid Viability
Polymerase Chain Reaction (RV-PCR) Protocol for
Detection of Yersiniapestis in Water Samples
National Homeland Security Research Center
Office of Research and Development
United States Environmental Protection Agency
Cincinnati, OH 45268
and
Lawrence Livermore National Laboratory
United States Department of Energy
Livermore, CA 94551
«*EPA
United Stakes
fcrtrircmmerttal Protection
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ii
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Disclaimer
U.S. Environmental Protection Agency
The United States Environmental Protection Agency through its Office of Research and
Development funded and managed the research described here (EPA IA DW-89-92328201-0). It
has been subjected to the Agency's review and has been approved for publication. 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 official EPA approval, endorsement, or
recommendation.
Lawrence Livermore National Laboratory
This document was prepared as an account of work sponsored by the Environmental Protection
Agency of the United States government under Contract DE-AC52-07NA27344. 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 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.
National Homeland Security Research Center
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
Nickel.Kathy@epa.gov or McCall.Amelia@epa.gov for assistance.
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Table of Contents
Disclaimer iii
U.S. Environmental Protection Agency iii
Lawrence Livermore National Laboratory iii
List of Tables vi
List of Figures vii
List of Abbreviations and Acronyms viii
Trademarked Products x
Acknowledgments xi
Executive Summary xii
1. Introduction 1
2. Materials and Methods 5
2.1 Bacterial Strains and Growth Conditions 5
2.2 Y. pestis Cell Suspension Preparation 5
2.3 Water Sample Types Used in This Study 6
2.4 Addition of Dust Background 6
2.5 Preparation of FeS04 and Humic Acid Solutions as Challenge Materials 6
2.6 Preparation of Dead Y. pestis Cell Suspensions 7
2.7 PCR Evaluation of Dead Y. pestis Cell Suspensions 8
2.8 Rapid-Viability PCR Method 8
2.9 Y. pestis C092 DNA Standards for Real-Time PCR 9
2.10 Real-Time PCR Analysis 9
2.11 Interpretation of RV-PCR Results 11
2.12 Data Analysis and Presentation 11
2.13 Estimation of DNA Copy Numbers and Cell Numbers from Real-Time PCR Results.... 12
2.14 Immunomagnetic Separation of Y. pestis Cells 13
2.15 Modified Filtration for Concentration of Y. pestis Cells 15
3. Quality Assurance and Quality Control 18
3.1 Laboratory Inspections 18
3.2 Calibration 18
3.3 Storage Conditions 18
3.4 Spiking 18
3.5 Real-time PCR Analysis 18
3.6 Replication 19
3.7 Controls 19
3.8 Data Quality Objectives/Data Quality Indicators 19
4. Results and Discussion 19
4.1 TASK 1: Incorporate DNA Extraction and Purification Steps into RV-PCR Protocol for Y.
pestis and Evaluate Protocol Parameters (Incubation Period, LOD) 20
4.1.1. Objectives 20
4.1.2. Overall Approach for Evaluating DNA Extraction and Purification Protocols for Y. pestis
Cells 20
4.1.3. Evaluation of Y. pestis-Specific Real-Time PCR Assays 20
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4.1.4. Comparison of Cell Number Estimated from PCR Results and Viable Cell Counts -
Evaluation of Modified Chemical Lysis (Promega MagneSil) Protocol 21
4.1.5. Comparison of PCR Results with Universal Reagents/Standard Cycling and Fast
Reagents/Fast Cycling Conditions 23
4.1.6. Evaluation of Heat Lysis Vs. Chemical Lysis for Y. pestis Cells 24
4.2 TASK 2: Further development and optimization of sample processing protocols for Y.
pestis cell recovery and growth 28
4.2.1. Objectives 28
4.2.2. Approaches Used for Y. pestis C092 Growth Optimization 28
4.2.3. Evaluation of Y. pestis Growth in IX BHI Broth in 48-Well Plate Format 28
4.2.4. Evaluation of Y. pestis Growth in IX BHI Broth (Prepared From 1 OX BHI) 30
4.2.5. Evaluation of Y. pestis Growth in 1XYPEB Compared to IX BHI Broth Prepared From 10X
BHI Broth 31
4.2.6. Growth of Y. pestis in 48-Well Plates and RV-PCR Analysis With Different To andTf
Aliquot Volumes 31
4.2.7. Modified Filtration for Concentration of Y. pestis Cells from Larger Volume Water
Samples 34
4.2.8. Evaluation of Immunomagnetic Separation for Concentration of Y. pestis Cells from
Larger Volume Water Samples 37
4.3 TASK 3: Further development and optimization of RV-PCR protocols for Y. pestis 40
4.3.1. Objectives 40
4.3.2. Evaluation of RV-PCR Method Performance with Complex Water Samples 40
4.3.3. Evaluation of RV-PCR Method Performance in a Dead Y. pestis Cell Background 47
5.0 Conclusions 55
6.0 References 57
Annex 1 Standard Operational Procedure - LLNL Manual Protocol for Rapid Viability
Polymerase Chain Reaction (RV-PCR) for Analysis of Yersinia pestis in Water Samples
59
Trademarked Products 60
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List of Tables
Table 1 Nucleotide Sequences* of the Primer/Probe Sets Used for Y pestis RV-PCR
Analysis 11
Table 2 Real-time PCR Results for Y. pestis C092 Genomic DNA Dilutions With Assays
or pPCPl and pMTl Plasmids and the Chromosome 21
Table 3 Comparison of Estimated CFU/mL from Real-Time PCR Analysis* (YC2 Assay)
With CFU/mL from Culture Analysis (With IX YPEB Prepared From 10X) 22
Table 4 Thermal Cycling Parameters for the Different Real-Time PCR Configurations 23
Table 5 Real-time PCR Results for the Plasmid Assays YpPl (pPCPl) and YpMTl (pMTl)
Using Fast/Fast and Universal/Standard Conditions With Y pestis DNA Standards 23
Table 6 Real-time PCR Results for the YC2 (Chromosomal) Assay Using Fast/Fast and
Universal/Standard Conditions With Y pestis DNA Standards 24
Table 7 Real-time PCR Results for DNA Extracted from Y pestis Cells by Heat or Chemical
Lysis (followed by Promega Kit Purification) and Analyzed by Chromosomal (YC2)
and Plasmid Assays (YpPl and YpMTl) - First Replicate Experiment 26
Table 8 Real-time PCR Results for DNA Extracted from Y pestis Cells by Heat or Chemical
Lysis (followed by Promega Kit Purification) and Analyzed by Chromosomal (YC2)
and Plasmid Assays (YpPl and YpMTl) - Second Replicate Experiment 27
Table 9 Growth of Y. pestis Cells in 48-Well Plates (3 mL IX BHI)* for -6 - 6 x 103
Colony Forming Units (CFU)/mL Starting Y pestis Cell Concentrations 29
Table 10 Growth of Y. pestis Cells in 48-Well Plates (3 mL BHI Prepared Using 10X BHI)*... 30
Table 11 Growth of Y. pestis Cells in 48-Well Plates (3 mL IX YPEB)* 31
Table 12 Effect of Time Point Aliquot Volume (250 and 500 |iL) on ACt for RV-PCR
Analysis: Y. pestis Cells in YPEB (Prepared Using 10X YPEB)* 33
Table 13 Real-time PCR Results for RV-PCR Analysis* of Y. pestis C092 pgm Cells
(~4 x 104) Collected by a Modified Filtration Approach 36
Table 14 Real-time PCR Results from RV-PCR Analysis of Water Samples Containing
Different Levels of Y pestis C092 pgnT Cells Processed by IMS - YC2 Assay 38
Table 15 Real-time PCR Results from RV-PCR Analysis of Water Samples Containing
Different Levels of Y pestis C092 pgnT Cells Processed by IMS - YC2 Assay
(Replicate Experiment) 39
Table 16 Real-time PCR Results for RV-PCR DNA Extracts from -180 Y. pestis C092 Cells
Added to 48-Well Plates With and Without Chemical or Biological Backgrounds 43
Table 17 Real-time PCR Results for RV-PCR DNA Extracts from -18 Y. pestis C092 Cells
Added to 48-Well Plates With and Without Chemical or Biological Backgrounds 44
Table 18 Real-time PCR Results for RV-PCR DNA Extracts from -100 Y. pestis C092 Cells
Added to 48-Well Plates With and Without Chemical or Biological Backgrounds 45
Table 19 Real-time PCR Results for RV-PCR DNA Extracts from -10 Y. pestis C092 Cells
Added to 48-Well Plates With and Without Chemical or Biological Backgrounds 46
Table 20 PCR Analysis of Components from Generation of IPA-Killed Cell Suspensions to
Assess DNA Content or Loss 49
Table 21. RV-PCR Results for 10- and 100-Cell Levels (Live Y. pestis Cells) With Different
IPA-Killed Target Cell Concentrations - 10-Fold Diluted DNA Extracts 51
Table 22 RV-PCR Results for 10- and 100-Cell Levels (Live Y. pestis Cells) With Different
IPA-Killed Target Cell Concentrations - YC2 Assay With 10-Fold Diluted DNA
Extracts (Replicate Experiment) 53
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Table 23 RV-PCR Results for 10- and 100-Cell Levels (Live Y. pestis Cells) With Different
IPA-Killed Target Cell Concentrations - YC2 Assay With Undiluted DNA Extracts
(Replicate Experiment) 54
List of Figures
Figure 1 Example real-time PCR response curves showing parameters (Ct [To], Ct [Tf], and
ACt) used in determining presence or absence of viable spores in the original sample 2
Figure 2 Comparison of the RV-PCR method vs. the traditional culture method for Y pestis 3
Figure 3 Flow chart for RV-PCR analysis of Y pestis cells from water samples 4
Figure 4 Pathatrix Immunomagnetic Separation system (Life Technologies, Inc.) 14
Figure 5 Flow chart for IMS-treated and control Y pestis cell suspensions 15
Figure 6 Flow chart for sample processing using the modified filtration approach followed
by RV-PCR analysis for Y pestis cells 17
Figure 7 Outline of Protocol Steps for Chemical Lysis (Promega MagneSil kit) and Heat
Lysis Procedures for DNA Extraction and Purification 25
Figure 8 Flow chart for generation and analysis of IPA-killed cell suspensions 48
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List of Abbreviations and Acronyms
Ab antibody
ABI Applied BioSystems, Inc.
ATD Arizona Test Dust
Ave average
B. anthracis Bacillus anthracis
BHI brain heart infusion
BHQ Black Hole Quencher
bp base pair
BSC biosafety cabinet
BSL-3 BioSafety Level-3
°C degrees Centigrade
CDC Centers for Disease Control and Prevention
CFU colony-forming units
CRP Critical Reagents Program
Ct cycle threshold
Ct (To) or To Ct Ct value at time zero (pre-incubation)
Ct (Tf) Ct value at time final (post-incubation)
Ct (T12) or T12 Ct Ct value after 12 hours incubation
Ct (T24) or T24 Ct Ct value after 24 hours incubation
Ct (T40) or T40 Ct Ct value after 40 hours incubation
ACt delta cycle threshold
DD distilled, deionized
DE diatomaceous earth
DNA deoxyribonucleic acid
DOE Department of Energy
dNTP deoxynucleotide triphosphate
dsDNA double-stranded DNA
ERLN Environmental Response Laboratory Network
EPA Environmental Protection Agency
F. tularensis Francisella tularensis
FAM 6-carboxyfluorescein
fg/[xL femtogram per microliter
FDA Food and Drug Administration
FERN Food Emergency Response Network
g gram
hr hour(s)
IMS immunomagnetic separation
ISO International Organization for Standardization
IP A isopropanol
kb kilobase
LLNL Lawrence Livermore National Laboratory
LOD limit of detection
LRN Laboratory Response Network
Hg microgram
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Hg/L micrograms per liter
Hg/mL micrograms per milliliter
|j,m micrometer
Mb mega base pairs
MF modified filtration
mg milligram
MG Miracle Gro®
min minute
|j,L microliter
mL milliliter
mg/L milligrams per liter
mm millimeter
mM millimolar
NA not applicable
NDT non-detect
ng/[jL nanograms per microliter
NHSRC National Homeland Security Research Center
NIST National Institute of Standards and Technology
nm nanometer
NTC No-Template Control
OD600 optical density at 600 nm
ORD Office of Research and Development
OW Office of Water
PBS phosphate buffered saline
PCR polymerase chain reaction
pg picogram
PMP paramagnetic particles
ppm parts per million
PI Principal Investigator
QA quality assurance
QC quality control
RCF relative centrifugal force
RNA ribonucleic acid
ROX 6-carboxyl-X-rhodamine
rpm revolutions per minute
RV rapid viability
RV-PCR rapid viability-polymerase chain reaction
SAP superabsorbent polymers
SD standard deviation
sec second
To time 0, prior to incubation
T: after 2 hr of incubation
T9 after 9 hr of incubation
T12 after 12 hr of incubation
T24 after 24 hr of incubation
T40 after 40 hr of incubation
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T48 after 48 hr of incubation
Tf time final, after incubation
TBA Tryptose Blood Agar base
TNTC too numerous to count
UNG uracil-N-glycosilase
UV ultraviolet
WLA Water Laboratory Alliance
Y pestis Yersinia pestis
YPEB Y pestis Enrichment Broth
IX 1-fold concentrated (no concentration)
10X 10-fold concentrated
Trademarked Products
Trademark
Holder
Location
ABI Gold™
Life Technologies
Carlsbad, CA
AB Applied BioSystems™
Life Technologies
Carlsbad, CA
AeraSeal™
Excel Scientific
Victorville, CA
AmpliTaq Gold®
Life Technologies
Carlsbad, CA
Autovials™
GE Healthcare
Noblesville, IN
Bacto™
Difco Laboratories
Franklin Lakes, NJ
Black Hole Quencher®
Biosearch Technologies
Petaluma, CA
Difco™
Becton Dickinson
Dynamag™
Life Technologies
Carlsbad, CA
Epicentre®
Epicentre Biotechnologies Inc.
Madison, WI
Invitrogen®
Life Technologies
Carlsbad, CA
Life Technologies™
Life Technologies
Carlsbad, CA
LIVE/DEAD® BacLight™
Life Technologies
Carlsbad, CA
MagneSil® Blood Genomic
Promega
Madison, WI
MasterPure®
Epicentre Biotechnologies
Madison, WI
Millipore®, Milli-Q™
Millipore Corp.
Billerica, MA
MicroFunnel™
Pall Corp.
Ann Arbor, MI
Miracle Gro®
The Scotts Company
Maryville, OH
Pathatrix®
Life Technologies
Carlsbad, CA
PicoGreen®
Life Technologies
Carlsbad, CA
Quant-iT™
Life Technologies
Carlsbad, CA
Qubit®
Life Technologies
Carlsbad, CA
TaqMan®
Life Technologies
Carlsbad, CA
Whatman®
GE Healthcare, Life Sciences
Pittsburgh, PA
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Acknowledgments
This work was performed under the auspices of the U.S. Department of Energy by Lawrence
Livermore National Laboratory (LLNL) under Contract DE-AC52-07NA27344. Funding for this
research was provided by the U.S. Environmental Protection Agency's (EPA's) National
Homeland Security Research Center (NHSRC).
Research Team
Lawrence Livermore National Laboratory
Staci Kane, Teneile Alfaro, and Anne Marie Erler
EPA Technical Lead
U.S. EPA National Homeland Security Research Center
Sanjiv Shah
Project Plan Reviewers
EPA National Homeland Security Research Center
Tonya Nichols
Technical Reviewers
EPA National Homeland Security Research Center
Gene Rice, Worth Calfee, and Tonya Nichols
EPA Office of Water - Water Security Division (OW)
Latisha Mapp
EPA Office of Chemical Safety Pollution Prevention - Office of Pesticide Programs Microbiology
Laboratory
Jafrul Hasan
Quality Assurance Reviewers
EPA National Homeland Security Research Center
Eletha Brady-Roberts and Ramona Sherman
Edit Reviewer
EPA National Homeland Security Research Center
Marti Sinclair, Alion Science and Technology, NHSRC Contract GS35F4594G
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Executive Summary
The U.S. Environmental Protection Agency (EPA) decision makers will need timely and reliable
water sample analysis results during the response to and recovery from a plague incident resulting
in contamination of the water infrastructure regardless of whether the contamination is a result of
a natural outbreak, laboratory accident, or a criminal or terrorist incident. It is known that
vegetative bacterial pathogens such as Yersiniapestis, the bacteria that cause plague, may remain
viable and infectious for some time in certain environments including water. Due to the historical
usage of Y pestis as a biological weapon and the occurrence of natural plague outbreaks, there is
a need for rapid and sensitive analytical methods for detection of viable Y pestis. The EPA Office
of Water (OW) is responsible for protecting and managing water resources. In this research effort,
the Rapid Viability Polymerase Chain Reaction (RV-PCR) method was developed for use by the
Water Laboratory Alliance (WLA), a network of laboratories for water sample analysis established
by the OW. The WLA is also a significant component of the EPA's Environmental Response
Laboratory Network (ERLN).
The RV-PCR method, which combines high throughput sample processing, short-incubation broth
culture, and sensitive, specific real-time PCR analysis before and after sample incubation, can
afford rapid and high-sensitivity detection of viable biothreat agents even in backgrounds of high
levels of debris, non-target microbial cells/spores, and dead target agent. In partnership between
the scientists at EPA's National Homeland Security Research Center (NHSRC) within the Office
of Research and Development (ORD) and the scientists at the Lawrence Livermore National
Laboratory (LLNL), the RV-PCR method was developed and optimized to meet the need for a
rapid analytical method for Y pestis detection. This method may serve as a model for other
vegetative bacterial pathogens of concern.
In this research effort, the RV-PCR method was developed and evaluated for Y pestis cells with
the following key features: 24-hr incubation (shortened from preliminary 48-hr incubation);
sample incubation in 48-well plates for high throughput culture and sample handling; sensitive,
10-cell level (10-99 cells) limit of detection (LOD); and robust sample processing steps that
accommodate complex sample backgrounds. By optimizing the cell processing procedure and
incorporating a deoxyribonucleic acid (DNA) extraction/purification procedure (based on
Promega Magnesil® reagents), a shorter incubation period was demonstrated with maintenance of
the 10-cell level LOD. These reagents showed good DNA yield and quality and are compatible
with automated processing, although other existing manual or automated platforms could be used
as well. Furthermore, improvements in the culturing step enabled reproducible growth even at low
inoculum levels (<10 cells per mL). Additional recommendations were made based on challenge
testing with potential soluble and insoluble chemical interferences, and live, non-target or dead
target biological interferences, addressing a range of potential "real world" complex sample types.
These included extending the incubation time to 36 hr to further reduce the method false negative
rate for samples with high dead target cell backgrounds (> 104 cells/mL). This effort also included
a preliminary investigation that showed RV-PCR was compatible with front-end methods to
concentrate cells from larger volume water samples such as immunomagnetic separation (IMS)
using magnetic beads coated with Y pestis-specific antibodies and a modified filtration approach
using superabsorbent polymers to maintain moisture in filtration devices. However, both of these
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approaches had their inherent limitations, which could challenge their operational use. While
outside the scope of this effort, RV-PCR is expected to be compatible with ultrafiltration for cell
concentration since a 10-cell level LOD was observed even in complex samples containing
chemical and biological interferences.
The RV-PCR method for detection of viable Y. pestis from water samples will help enhance the
WLA capability for rapid, reliable, and high throughput sample analysis in case of a natural
outbreak, laboratory accident, or a criminal or terrorist incident of plague.
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1. Introduction
The U.S. Environmental Protection Agency (EPA) is designated as the lead federal agency during
the remediation phase of a response to a bioterrorism attack. EPA is also designated as the Sector-
Specific Agency for water and wastewater systems. Decision makers will need timely and reliable
water sample analysis results during biological agent response and recovery efforts. Y pestis is one
of these agents which could be introduced into water infrastructure due to a natural outbreak,
laboratory accident, or intentional contamination. It is known that vegetative bacterial pathogens
such as Yersinia pestis (Y. pestis, the causative agent of plague) may remain viable and infectious
for some time in certain environments including water. Due to the historical usage of Y pestis as
a biological weapon and the occurrence of natural plague outbreaks, there is a need for rapid and
sensitive analytical methods for detection of viable Y pestis. Within EPA, the Office of Water
(OW) is responsible for protecting and managing water resources. The OW has established the
Water Laboratory Alliance (WLA), a network of laboratories for water sample analysis. The WLA
is also a significant component of the EPA's Environmental Response Laboratory Network
(ERLN). The WLA needs rapid and reliable sample analysis methods to assess the presence of live
Y pestis.
To help meet this need, the National Homeland Security Research Center (NHSRC) within the
EPA's Office of Research and Development (ORD), in collaboration with the Lawrence Livermore
National Laboratory (LLNL) of the Department of Energy (DOE), has developed and optimized
the Rapid Viability Polymerase Chain Reaction (RV-PCR) method for rapid detection of viable Y
pestis in water samples. This method can serve as a template for the detection of other vegetative
bacterial pathogens, where modifications can be made for differences in growth requirements and
characteristics.
The current culture-based methods used for Y pestis detection are labor-intensive and low
throughput such that only 30-40 samples can be processed per day per laboratory, with confirmed
results obtained days later. More rapid viability methods are needed as part of the EPA's
capabilities to ensure public safety and to help mitigate impacts of facility and infrastructure
closures following a biological agent release. It is well understood that rapid detection methods
such as real-time polymerase chain reaction (PCR) cannot distinguish between live (potentially
infectious) and dead pathogens; however, features of real-time PCR were leveraged for
development of RV-PCR. The RV-PCR method combines high throughput sample processing,
short-incubation broth culture, and sensitive, specific real-time PCR analysis before and after
sample incubation, to detect low concentrations of viable bacterial threat agents. Specifically, the
method uses the change in real-time PCR response, referred to as the change in cycle threshold
(Ct) or ACt, between the initial (before sample incubation) Ct at time 0 (Ct To) and the final Ct
after incubation (Ct Tf). Example PCR response curves are shown in Figure 1 along with the
criteria for positive detection, namely ACt > 6. The method allows detection of viable target
biothreat agent even in backgrounds of high levels of debris, non-target microbial cells/spores, and
dead target agent.
The RV-PCR method for Y pestis detection not only generates rapid results but also can provide
a higher throughput capability as compared to the traditional culture-based methods, and hence,
increases the laboratory capacity for sample analysis. In place of multiple sample dilutions, several
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growth media agar plates, and enrichment culture per sample used by the culture method, the RV-
PCR method uses a single well per sample on a 48-well plate ( Figure 2).
Time- 0
response,
CT (To)
¦ PCR analyses before (T0) and after (Tf)
incubation
¦ A shift in PCR Ct value indicates an
increase in DNA, which is itself due to
an increase in cell number due to
organism viability
¦ The method accurately distinguishes
live cells from dead cells based on CT
(T0), CT (Tf) and ACT
¦ For a positive result,
ACT=(CT[T0]-CT[Tf])>6
500
0
1 400
0
w>
1 300
200
PCR Cycle
Endpolnt
response,
CT (Tf)
Figure 1 Example real-time PCR response curves showing parameters (Ct [To], Ct [Tf],
and ACr) used in determining presence or absence of viable spores in the original sample.
A significant shift in PCR response curve indicates an increase in DNA and thus, cell number. A
curve is shown for the Time 0 (TO) response, however, if no PCR response is observed there
would be a flat line and the CT would be set to the total number of cycles used (e.g., 45) in order
to calculate a ACt value.
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RV-PCR Method
1 sample -> 1 well on 48-well plate 2 PCR analyses
Multiple 48-well plate in one incubator
Confirmed results in < 36 hr
Plating Method
46 Samples + Controls
On one 48-Well Plate
Multiple 48-Well Plates
in One Incubator
1 sampled 11 culture plates + culture tube
presumptive Y. pestis colonies 2-5 colonies for PCR analyses/sample
Confirmed results in ~ 72+ hr
Serial dilution and plating
Enrichment culturing
Filtration and plating
Multiple Incubators
Figure 2 Comparison of the RV-PCR method vs. the traditional culture method for Y.
pestis.
This effort significantly expanded a previous effort where LLNL and NHSRC scientists developed
preliminary RV-PCR protocols for Y. pestis and Francisella tularensis (/•'. tularensis). The project
work conducted under Interagency Agreement # DW-89-92243001-0, led to protocols for Y. pestis
and /•'. tularensis cells from wet wipes and buffered water samples (US EPA Internal Report,
2010). Results from the previous effort showed the potential for higher throughput sample
processing in 48-well plates and shorter incubation periods for confirmation of viable pathogen
presence compared to current traditional culture-based methods; these efforts were the starting
point for the current effort described here.
This project focused on the water matrix with chemical and microbial challenges. Chemical
challenges included ferrous sulfate, humic acids, and metal oxides present in Arizona Test Dust
(ATD). These materials could negatively affect cell growth and cell recovery from water samples
and/or interfere with subsequent analysis. Microbial challenges included dead Y. pestis cells and
microorganisms present in non-autoclaved ATD including Bacillus spp. and other non-target
bacteria as well as fungal species (Rose et al„ 2011). The virulent Y. pestis C092 reference strain
was used for protocol development. In addition, the attenuated Y. pestis C092 pgm strain was
used in specific cases as identified in the report; in particular, this strain was used in evaluation of
the immunomagnetic separation (IMS) and modified filtration (MF) approaches as well as
generation of a dead cell background for RV-PCR method evaluation. In this effort, DNA
concentration and purification steps were incorporated to further shorten the timeline. Features of
real-time PCR analysis that benefit rapid analysis include low detection limits (typically <10 DNA
copies per reaction), several order of magnitude linear range (~8 logs), and ability to detect low
numbers of target organisms in the presence of high populations of non-target organisms; whereas,
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traditional culture methods are challenged with environmental backgrounds where target bacteria
may be outcompeted by indigenous microorganisms.
The RV-PCR protocol steps and some of the equipment for Y. pestis are shown in Figure 3. The
manual RV-PCR protocol (without cell concentration) developed in this study could be readily
automated for higher throughput since the same materials and procedures would be used. In
addition, automated platforms for DNA extraction and concentration are often available in
laboratories including those in the Centers for Disease Control and Prevention Laboratory
Response Network (CDC LRN).
Add 2.7 mL to
48-well plate
Water
Sample
Add 10X growth
medium
Mix; Take T24
aliquot for PGR
DNA extraction
& purification
T24 aliquot
PGR analysis
DNA extraction
& purification
T0 aliquot
PGR analysis
Viable cells
present based on
ACt and CT (T24)
Incubate 30°C
24 hr
Mix; Take T0
aliquot for PGR
Figure 3 Flow chart for RV-PCR analysis of Y. pestis cells from water samples. Using a 48-
well plate, up to 3.5 milliliter (mL) total volume can be used per 5-mL well, such as 2.7 mL
water sample with 0.3 mL 10X broth (as shown) or up to 3.15 mL water sample with 0.35 mL
10X broth.
This report describes experiments and results focused on three major tasks:
Task 1. Incorporate DNA extraction/purification procedure into Y. pestis RV-PCR
protocol
Task 2. Further develop and optimize the sample processing procedure for Y. pestis
cell recovery and cell growth
Task 3. Further develop and optimize the RV-PCR protocol for Y. pestis
Tasks 1 and 2 included development and optimization of procedures for Y. pestis culturing and
DNA recovery within the RV-PCR protocol and Task 3 used the optimized procedures for
evaluation of the entire RV-PCR protocol. Challenge testing for Task 3 included the following
additions: (i) humic acid and ferrous sulfate as potential chemical interferents; (ii) live (native)
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Water samples
48-well plate w/breathe seal
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ATD as a source of potential growth-competing microorganisms (non-target cells/spores) and
metal oxides; and (iii) dead target cells as background for either post-decontamination or natural
degradation scenarios.
With the RV-PCR method developed in this effort, Y. pestis Enrichment Broth (YPEB) diluted
from 1 OX to IX concentration with the water sample was shown to be optimal for Y pestis growth
in the 48-well format. The 10-cell level (10-99 cells) limit of detection (LOD) was observed after
24 hr incubation even in the presence of soluble chemicals, insoluble particulates, and non-target
cells and spores. Based on the results, a longer incubation period of 36 hr was recommended for
samples containing high concentrations of dead Y pestis cells (>104/mL). The RV-PCR method
enabled higher throughput and shorter time to results (-36 hr for 48 samples and controls with a
24-hr incubation period) compared with traditional culture methods.
2. Materials and Methods
2.1 Bacterial Strains and Growth Conditions
In this effort, the pathogenic Y pestis C092 strain and the attenuated Y pestis C092 pgm strain
were used. The pgnT strain lacks the 102-kilobase (kb)pgm locus, which contains a pigmentation
section and a high pathogenicity island with virulence genes (Buchrieser et al., 1999). The two
strains are from the LLNL strain collection and were verified by performing real-time PCR
analysis on genomic DNA using primers and probes specific to the Y pestis chromosome and
plasmids. Although the pgnT strain has a deleted region, none of the assays used in this effort
targeted this region so all three assays also detected this strain.
The Y pestis strains were initially grown in Brain Heart Infusion (BHI) broth (prepared from
dehydrated powder; Becton Dickinson, Cat. No. 237500) at 28 degrees Centigrade (°C) and on
BHI agar plates (with Bacto™ Agar) at 28°C; however, during this study, it was determined that
improved cell growth occurred with plates prepared from Tryptose Blood Agar base without blood
(TBA; Becton Dickinson, Cat. No. 223220) and Y. pestis Enrichment Broth (YPEB) (Doran et al.,
2013) rather than BHI broth. Therefore, TBA and YPEB were used for the majority of the
experiments (Please see the SOP).
In addition, 30°C was used for some subsequent experiments where noted. Frozen stocks (-80°C)
were prepared with 10% glycerol and used to start cultures on TBA plates for experiments. Two
different types of solid agar plates were used for the study. Initially BHI plates were used; however,
since inconsistent results were observed within and between experiments, a recommendation was
made to use TBA (without blood). TBA plates led to high cell counts relative to BHI plates as well
as more consistent results (less variability between replicate plates).
2.2 Y. pestis Cell Suspension Preparation
For each experiment, cells were propagated starting with agar plates (BHI or TBA) that were
streaked from -80°C glycerol stocks. Plates were incubated at 28°C for 2 to 4 days prior to selecting
2-3 colonies with similar morphology for inoculating 5-mL liquid cultures in 50-mL conical tubes.
For initial experiments with BHI broth, successive overnight (18-26 hr) 5-mL cultures were used
5
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in each case starting with an optical density at 600 nanometer (nm) (OD600) of-0.1 (adjusted by
dilution with BHI), which corresponds to approximately 6-7 x 106 CFU/mL. After three overnight
cultures, cells were harvested by centrifugation (3,100 relative centrifugal force [RCF] at 4°C for
15 min) and suspended in BHI directly for experiments testing IX BHI and suspended in PBS for
experiments in which one part 10X (10-fold concentrated) BHI was added to nine parts cell
suspension in PBS (referred to as IX BHI prepared from 10X BHI).
Since YPEB provided more consistent growth, the majority of experiments used a 5-mL YPEB
overnight culture with incubation at 28-30°C. The cells were prepared as described for culturing
in BHI broth except IX YPEB was used directly or 10X YPEB was added to cells in PBS to bring
the final concentration to IX YPEB. Cells were then diluted in IX YPEB or PBS to OD600 -0.1,
and ten-fold serial dilutions were performed in IX YPEB or PBS buffer to achieve the desired
starting cell density (CFU/mL) in three mL final volume per well of a 48-well plate.
2.3 Water Sample Types Used in This Study
PBS (Teknova, Inc., Hollister, CA; Cat. No. P0261) was used as a reference matrix because it
maintained cell viability and represented a reproducible matrix, in terms of chemical composition
and pH for the method evaluation. Throughout the report, the term "sample" refers to Y pestis cell
suspensions prepared in PBS. Materials were added to this buffer including i) iron sulfate and
humic acid (Sigma-Aldrich, Cat. No. 53680-10G) to represent chemical interferences, ii) ATD
(Section 2.4) to represent chemical, biological (live, non-target microorganisms), and physical
challenges, and iii) dead Y pestis cells to assess the background effect for post-decontamination
applications.
2.4 Addition of Dust Background
ATD (ISO 12103-1, A3 Medium Test Dust; Powder Technology, Arden Hills, MN) was used to
evaluate biological and chemical inhibition effects on Y pestis growth and PCR. Chemical
composition analysis performed by the manufacturer indicated the material consisted of: SiC>2 (68-
76%), AI2O3 (10-15%), Fe203 (2-5%), Na20 (2-4%), CaO (2-5%), MgO (1-2%), Ti02 (0.5-
1.0%), and K2O (2-5%). Dust was added at 4 mg/mL. Microbiological analysis showed that the
dust had -5 x 104 CFU/10 mg background microbes including fungi and bacterial spores (Rose et
al., 2011). Dust was non-sterilized, made into a slurry, and added to samples at a final
concentration of 4 mg/mL.
2.5 Preparation of FeS04 and Humic Acid Solutions as Challenge Materials
Iron sulfate (heptahydrate; Sigma-Aldrich, Cat. No. 215422) and humic acid (Sigma-Aldrich Cat.
No. 53680-10G) were added to samples to test for chemical interferences effecting Y pestis growth
and PCR. Humic acid was used as a surrogate for natural organic matter. An FeSC>4 solution was
prepared in sterile distilled, deionized (DD) water and added to samples at a final concentration of
10 microgram (p,g)/mL Fe2+. A humic acid solution was also prepared in sterile DD water and
added to samples at a final concentration of 50 [j,g/mL. These concentrations were within the range
of values expected for drinking water samples (NRC, 1979; WHO, 1996; US EPA, 2005) although
they represented the higher end of the range.
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2.6 Preparation of Dead Y. pestis Cell Suspensions
Different killing methods were evaluated to generate intact, dead cells that did not lose their DNA
upon disinfection. Initially, UV irradiation and desiccation were proposed; however, from previous
studies it was known that these methods were not very reproducible and for desiccation, long time
periods (e.g., 2-4 weeks) were required to produce complete and nearly complete disinfection of
Y pestis cells (Staci Kane, personal communication). Therefore in this effort, autoclaving,
antibiotic exposure, and isopropanol (IPA) exposure were investigated. It was determined that
autoclaving led to DNA degradation so it was not used for generating a dead cell background. For
antibiotic treatment, doxycycline was selected since there have been no reports of resistance to this
antibiotic. In order to avoid generating a resistant pathogenic strain, the pgm strain was used.
Doxycycline was used at 160 |j,g/mL for 24 and 48 hr; this concentration is more than 100 times
that reported for the minimum inhibitory concentration (Hernandez et al., 2003). While the
antibiotic treatment showed that cell proliferation measured by spectrometry (OD600) was halted
after treatment, significant viable cells still remained when the suspension was harvested, washed
in PBS, and plated. Therefore, IPA was investigated since it is often used to generate dead cells as
negative controls for cell staining kits for viability analysis using microscopy or flow cytometry
(e.g., LIVE/DEAD® itocLight™ Bacterial Viability Kit, Life Technologies, Inc.).
For generation of IPA-killed cells, an overnight culture of Y pestis C092 pgnT was prepared.
From this culture, a 100 mL culture was started after dilution of cells to OD600 -0.01 in YPEB.
The culture was incubated at 30°C (with shaking at 180 revolutions per min [rpm]) until OD600
-0.3-0.4 was achieved. Cells were then split into four 20-mL aliquots in 50 mL conical tubes and
harvested by centrifugation (3,100 RCF at 4°C for 15 min). Pellets were then suspended in 6 mL
of PBS. For the cells treated with IPA, 14 mL of 99+% IPA were added to the 6-mL suspension
to yield a final IPA concentration of-70%. For the control treatment, 14 mL PBS were added. The
cell suspensions were mixed gently and incubated for 1 and 2 hr at room temperature. Suspensions
were gently mixed every 30 min. After incubation, the suspensions were centrifuged at 4,000 rpm
and 4°C for 15 min. The supernatant was removed and the pellets washed in 20 mL PBS once
more followed by centrifugation, removal of supernatant and final suspension in PBS to 20 mL.
During washes and preparation of final cell suspension, the fractions were retained for subsequent
PCR analysis as outlined below.
The IPA-killed cell suspensions were divided into 1-mL aliquots for storage at 4°C until use, with
one aliquot used for each experiment. The calculated concentration based on the controls processed
in parallel was -4.1 x 107 per mL. The IPA-killed aliquots were used over a 40-day period and in
each case the cells were tested by PCR prior to use to ensure that DNA was not leaking from the
cells. Testing included heat lysis of both the original IPA-killed suspension and the cell pellet after
centrifugation of 0.5 mL and removal of 300 [xL supernatant (as for RV-PCR sample analysis).
Heat lysis was conducted at 95°C for 5 min followed by placement on ice for 2 min, centrifugation
(20,800 RCF at 4°C for 5 min), and removal of liquid for PCR analysis (leaving cell debris pellet
in tube). An aliquot from the 300 [iL supernatant was also analyzed by PCR to determine if
significant DNA was lost during this step, or the DNA largely remained with the cell pellet. The
original cell suspension prior to IPA treatment as well as the control treatments (treated with PBS
instead of IPA) were serially diluted and plated onto TBA plates for quantitation. For experiments
with different concentrations of dead cells, the stock suspension was diluted with PBS to achieve
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the desired dead cell level based on plate counts from control processed in parallel with IPA-killed
cells.
2.7 PCR Evaluation of Dead Y. pestis Cell Suspensions
PCR analysis was used to determine whether dead cells remained intact and DNA remained inside
the cells. During cell suspension preparation, washed cells and supernatants were analyzed. For
the initial supernatant following IPA treatment and centrifugation, 10- and 50-fold dilutions were
analyzed to attempt to dilute out the IPA and obtain PCR data. It was important to evaluate how
much DNA may have been lost during each processing step.
In addition, prior to using stored IPA-killed cells for an experiment, the suspension was centrifuged
at 20,800 RCF at 4°C for 5 min. The supernatant was used to test for DNA loss by PCR analysis
with the YC2 assay. In addition, the remaining cell pellet was lysed by heat and analyzed by PCR.
The original IPA-killed cell suspension was also subjected to heat lysis and PCR analysis. Results
from the supernatant, heat-lysed cell pellet, and heat lysed IPA-killed cell suspension were
compared to determine if DNA would be lost during RV-PCR sample processing and analysis
(which includes an additional centrifugation step). The YC2 assay was used following the
conditions outlined in Section 2.10.
2.8 Rapid-Viability PCR Method
An outline of the RV-PCR protocol steps is shown in Figure 3, including pictures of some of the
equipment used in sample processing and analysis. In contrast to the RV-PCR protocol for B.
anthracis spores, filtration could not be used to concentrate vegetative cells and reliably maintain
their viability. Therefore, the water sample was not filtered but rather was prepared using 10X-
concentrated broth, in the proper ratio, in order to use as much of the sample as possible and still
provide optimal growth conditions. The wells of the 48-well plates accommodate 5 mL such that
up to 3.15 mL water sample and 0.35 mL 10X broth could be used. In this study 2.7 mL sample
was added to 0.3 mL 10X broth.
After mixing by pipettor, a 0.5 mL aliquot was removed from the total 3.0 mL were withdrawn
from each well before incubation (To aliquot), transferred to 2 mL Eppendorf tubes, and
centrifuged at 20,800 RCF for 10 min at 4°C, after which 300 [xL were removed. Pellets in the
remaining 0.2 mL were then frozen prior to DNA extraction and PCR analysis following the
protocol detailed below. During method development, in some cases (as specified in the report),
0.25 mL aliquots were removed and processed as described above except that only 50 [xL were
removed, leaving 0.2 mL pellets. The 48-well plate was sealed with a sterile AeraSeal™breathable
adhesive seal (Excel Scientific, Cat. No. BS-25) and incubated for different time periods from 12
to 40 hr at 28-30°C with shaking at 180 rpm prior to removal of the 0.5-mL aliquots (or 0.25 mL
aliquots as noted) for the different time points. Aliquots were typically stored at -20°C and
processed 1-2 days after receipt. However, samples could be processed immediately if staff were
working in shifts (e.g., 3, 8-hour shifts per day) to address sample volume and decrease time to
results. While manual processing was used in this study, automated DNA extraction protocols
could also be used.
Each 0.2 mL suspension (resuspended pellet) was processed for DNA extraction and purification
using the Promega paramagnetic particle (PMP)-based kit (MagneSil® Blood Genomic, Max Yield
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System; Promega, Cat. No. MD1360). The method was modified from that developed for B.
anthracis cells (US EPA, 2012), as described below. When used with the appropriate buffers, the
PMPs bind and later release DNA with appropriate buffers resulting in DNA concentration and
purification. The 0.2 mL aliquot was thawed and 800 |j,L of Lysis Buffer were added followed by
vortex mixing and incubation at room temperature for 5 min. Next, 600 |j,L of PMP Mix
(containing Lysis Buffer) were added and vortex mixed (modification). The tubes were placed on
magnetic rack and the PMPs were adhered to the side of the tube next to the magnet, and the
supernatant was removed by pipetting. One lysis wash step with 360 |j,L of Lysis Buffer was
included, followed by vortex mixing, placing on the magnetic rack, and subsequent supernatant
removal. Two washes with 360 |j,L of Salt Wash were then performed, in each case followed by
mixing by vortexing and removal of the supernatant. Finally, two washes with 500 |j,L of Alcohol
Wash solution were performed with mixing by vortexing and supernatant removal. A final wash
with 70% ethanol was included to enhance PMP drying. PMPs were air-dried for 2 min and then
dried at 80°C for 20 minutes. DNA was then eluted by addition of Elution Buffer followed by
cycles of vortexing and heating at 80°C. Samples were allowed to cool for 5 min prior to mixing
and transferring to the magnetic rack. Typically 170-200 |j,L were recovered and transferred into
a clean 1.5 mL Eppendorf tube. If particles remained, the sample was subsequently centrifuged at
20,800 RCF for 5 min at 4°C, and the supernatant was transferred to a clean Eppendorf tube. The
DNA extracts were stored at -20°C until they could be analyzed by real-time PCR. Both undiluted
and 10-fold diluted DNA extracts (prepared in PCR water) for both To and later time points were
also analyzed by PCR to check for PCR inhibition (i.e., if the difference between Ct values for 10-
fold diluted and undiluted extracts is negative and/or significantly less than three).
2.9 Y. pestis C092 DNA Standards for Real-Time PCR
Y pestis C092 DNA standards were generated from harvested cells from overnight incubation of
5 mL YPEB cultures inoculated from 2-3 individual colonies from TBA plates. A MasterPure™
Complete DNA and RNA (ribonucleic acid) Purification Kit (Epicentre® Biotechnologies Inc. Cat.
No. MC85200) was used to extract genomic DNA following the manufacturer's protocol. This kit
is designed for producing genomic DNA from a small number of larger volume cultures to generate
higher quantities of DNA, whereas, the Promega Magnesil kit, optimized for use in RV-PCR
(Section 2.8), is designed for a large number of small sample volumes (0.2 mL after concentration
via centrifugation). The resulting genomic DNA was measured using the high sensitivity Quant-
iT™ DNA assay (Invitrogen, Cat. No. Q32854) with a Qubit™ fluorometer (Cat. No. Q33216).
Standard concentrations prepared in PCR-grade water ranged from 1 nanogram (ng)/|jL to 1
femtogram (fg)/|jL. Each PCR plate contained seven 10-fold dilutions, ranging from 5 ng per 25-
[iL PCR to 5 fg per 25-|iL PCR.
2.10 Real-Time PCR Analysis
The foundation for RV-PCR assay development is sensitive and specific real-time PCR assays. In
a previous study, high-quality signatures developed by Dr. Sanjiv Shah (while at Edgewood
Chemical and Biological Center of the Department of Defense) and LLNL using computational
tools for primer and TaqMan® probe design were used to design Y pestis real-time PCR assays
(US EPA Internal Report, 2010). In addition, in silico analysis and rigorous wet-chemistry
screening approaches were used to further down-select candidate signatures, by screening against
an extensive panel of environmental extracts, bacteria, eukaryotes, near-neighbors, and target
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strain DNAs. Furthermore, the down-selected assays (from 52 candidates) were tested against 12
target DNA templates in order to yield sensitive assays targeting the chromosome (YC2 assay)
and the pMTl plasmid (PMT1 assay). In addition, an assay for pPCPl (EPA-YpPl, referred to as
YpPl for this study) developed by Dr. Shah met the stringent screening requirements and showed
excellent sensitivity in the previous effort. Therefore, these three assays were used in this effort to
optimize the RV-PCR protocol for detection of viable Y pestis from water samples.
An Applied Biosystems® 7500 Fast Real-Time PCR System was used to perform Real-time PCR.
Each well of a 96-well PCR plate contained five |xL sample aliquots added to 20 |xL of PCR mix.
While three assays were used as mentioned, the majority of analysis used the YC2 assay. This
enabled more accurate analysis of DNA recovery efficiency and comparison with culture data
since the copy number for the chromosome marker could be more accurately assumed to be one
per cell. This assumption was confirmed by analysis of the Y pestis C092 genome sequence.
The YC2 assay targets a hypothetical protein with similarity to the Bordetella pertussis BapA
protein and the Escherichia coli YchA protein. These are autotransporter proteins of a type V
secretion system and these systems have been linked to virulence in Gram-negative bacteria
(Derbise et al., 2010). The YpPl assay, also referred to as Yp-EPAl was developed by EPA (Sanjiv
Shah, personal communication) and targets the plasminogen activator/coagulase (pla) gene, which
plays a role in virulence. Finally, the YpMTl assay targets the caflR gene, a positive regulator of
the F1 operon (encoding the F1 capsule antigen involved in virulence).
The PCR mix contained TaqMan® 2X Universal PCR Master Mix (Life Technologies, Cat. No.
4304437), which includes AmpliTaq Gold® DNA polymerase, deoxynucleotide triphosphates
(dNTPs), a 6-Carboxyl-X-Rhodamine (ROX) passive reference dye (for signal normalization), and
AmpErase® UNG (uracil-N-glycosilase) which prevents carry-over contamination (from PCR
products). The mix also contained forward and reverse primers and a probe labeled at the 5' end
with FAM (6-carboxyfluorescein) for the reporter dye and labeled at the 3' end with Black Hole
Quencher® (BHQ-1) for the quencher dye. The assay primer and probe sequences are listed in
Table 1. PCR-grade water was used to make the mix volume up to 20 [j,L per reaction and 5 [xL of
sample were added to bring the total volume to 25 [xL. The following cycling conditions were
used: 2 min at 50°C for UNG incubation, 10 min at 95°C for DNA polymerase activation, and 45
amplification cycles (5 sec at 95°C for denaturation and 20 sec at 60°C for annealing/extension).
Three replicate samples were analyzed for experimental condition, and three replicate PCR
analyses were conducted per sample replicate. DNA extracts from different time points from the
same samples were analyzed on the same plate to standardize the analysis conditions. The ROX
dye in the ABI Universal Master Mix was used to normalize the fluorescent reporter signal.
Automatic baseline and threshold settings were used throughout.
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Table 1 Nucleotide Sequences* of the Primer/Probe Sets Used for Y. pestis RV-PCR
Analysis
Assay
Forward Primer
Reverse Primer
Probe
Amplicon
Length
(bp)
YC2
CAACGACTAGCCAG
GCGAC
CATTGTTCGCACG
aaacgtaa
TTTTATAACGAT
gcctacaacggc
tctgcaa
78
YpPl
tgggttcgggcaca
tgata
ccagcgttaatt
acggtaccataa
cttactttccgt
gagaagacatc
CGGCTC
101
YpMTl
ggtaacagattcgt
ggttgaagg
ccccacggcagt
ataggatg
TCCCTTCTACCC
aacaaaccttta
aaggacca
99
* Sequences are listed in 5' to 3' orientation, bp = base pair.
2.11 Interpretation of RV-PCR Results
As a starting point for RV-PCR detection of viable Y pestis cells, the criteria developed for B.
anthracis were employed; specifically for positive detection, the endpoint PCR Ct or Ct value at
time final (post-incubation), Ct (Tf) < 39 and the ACt (Ct [To] - Ct [Tf]) > 6 (where f = final) are
required. For initial optimization, most of the work was conducted with 24 hr incubation, such that
Tf = T24. For cases where no PCR response was obtained (non-detect results), the Ct values were
set to 45 (since 45 PCR cycles were used), in order to calculate ACt. A ACt > 6 represented an
increase in DNA concentration of approximately 2-log, as a result of the presence of viable cells
in the original sample that propagated during incubation. Depending on end user requirements, a
higher ACt (Ct [To] - Ct [Tf]) > 9 (approximately three log increase in DNA concentration), and
a corresponding lower end point (Ct of < 36) could be used. For individual replicates within an
experiment, the RV-PCR result was considered positive when at least 2 of 3 replicates met the
algorithm requirement.
The RV-PCR method LOD was equivalent to the Y pestis cell level where 100% of the spiked
samples had Ct (Tf) of < 39 with a ACt > 6. This was essentially an analytical LOD of the RV-
PCR method and did not take into account any losses that could occur from sampling and sample
handling prior to RV-PCR analysis.
2.12 Data Analysis and Presentation
The criteria for positive/negative detection was based on both ACt and the Tf Ct. Data tables show
both individual PCR replicates as well as averages and standard deviations calculated in Microsoft
Excel®. If a single PCR replicate was positive and the other two replicates were non-detect, the
sample was considered negative or non-detect (NDT) and the sample Ct was set to 45, in order to
calculate ACt. Single replicate positive high Ct values (e.g., 39-44) were likely due to cross
contamination.
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The overall SD from all sample replicates was calculated using the following equation,
Overall or joint SD = \ — l)s:2 -»• fn- — + {Jt5 — IJsg2 + (nj x [Xj — .?]" \ -t-
(«2 x [X2 - f]2) + (n5 x [X3 - .?]-»]/{«„ + n2 + «5 - i)}
where nv «2, and n3 = the number of PCR analyses per sample for sample replicates 1, 2, and 3;
sl3 s2, and s3 = the SD of the Ct values for the individual samples; Xv X2, and X3 = the average Ct
values for the individual samples; x= the overall average Ct value for the samples. The overall SD
equation was modified accordingly for either two or four replicate samples or positive controls.
In cases where three replicate PCR analyses per sample (or control) were conducted, the overall
average for the replicate samples (or controls) was simply the average of the individual sample (or
control) averages. Culture data are shown in CFU/mL or CFU/sample (corrected for dilution)
based on the average and SD of triplicate plates with colony counts within the range of 25-250
CFU/plate.
2.13 Estimation of DNA Copy Numbers and Cell Numbers from Real-Time PCR Results
Comparison was made between actual CFU/mL measured by plate counts and corrected for
dilution and estimated cell numbers from Real-time PCR analysis using the YC2 chromosomal
assay. The following equation was used to calculate fg/target (assuming one target per genome):
bp r mol bp . r 650 g ^ r 101S fg^_ fg
( —) x ( — ) x C ) =
V £ mo v m23 ' J \ ~ J
target (genome) 6.023 x 1023 mol bp g target
As an example for Y. pestis,
, 4.83 x 106 N , mol bp N , 650 g N , 101S fg N 5.21 fg
( ) X ( ) X ( — ) x ( — ) = —
target 6.023 x 1023 mol bp g target
Assumptions included that there was one genome copy per cell and that the number of base pairs
(bp) per genome copy was 4.83 x 106 (by adding the bp from one copy of the chromosome [4.65
mega base pairs, Mb] and one copy each of the three plasmids, pCDl [70.3 kb], pPCPl [9.6 kb],
and pMTl[96.2 kb]).
In order to estimate the CFU/mL from the PCR Ct value the following equation was used:
Ct = — m(logfg DNA) + b
where m is the slope and b is the y-intercept from the standard curve obtained when plotting Ct
vs. log fg DNA for genomic DNA concentrations ranging from 5 ng to 5 fg (10-fold dilutions).
The log fg DNA (per 5 (jL) was then converted to fg DNA per mL by multiplying with the
appropriate dilution factor. In order to convert fg DNA per mL to targets (or genome equivalents)
per mL, the value was multiplied by It was assumed that genome equivalents were equal
to cell equivalents (i.e., one genome copy per cell). The CFU per plate was also converted to CFU
per mL, based on the amount of the sample plated and the dilution plated. To obtain the log
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difference, the targets per mL and CFU/mL were converted to log values and subtracted from each
other. For the data shown, the log difference = log targets from PCR/mL - log CFU from
plating/mL.
2.14 Immunomagnetic Separation of Y. pestis Cells
Since large volume water samples may need to be processed for detection of Y pestis cells,
immunomagnetic separation (IMS) was investigated as a front-end concentration method upstream
of RV-PCR analysis. IMS has been used successfully to concentrate Y pestis cells from complex
food rinsates (Himathongkham et al., 2007; Amoako et al., 2012; Darcy Hanes, Food and Drug
Administration [FDA], personal communication) with both a Applied Biosystems™ Pathatrix®
Auto concentrator (Life Technologies) and an /CropTheBug system (Filtaflex, Inc., Almonte,
Ontario, Canada). In addition, IMS approaches have been used to capture bacterial spores from
soil (Laura Rose, CDC, personal communication). Figure 4 shows the Pathatrix instrument, which
uses a disposable syringe system for mixing the sample with the antibody-coated magnetic beads.
In this effort, a Pathatrix system was used to concentrate the initial suspension (up to 60 mL
although 33 mL was used in this case) to 0.1 mL solution containing cells bound to magnetic beads
conjugated with a Y pestis-specific antibody (330-fold concentration). For this effort, polyclonal
antibodies specific to Y pestis were obtained from the Critical Reagents Program (CRP) through
BEI Resources, Inc., Manassas, VA (Cat. No. AB-G-YERS). The protocol for bead conjugation
and Pathatrix operation from Life Technologies was followed.
It should be noted that the captured cells could be processed directly by culture, immunoassay, or
real-time PCR (following DNA extraction), or alternatively the cells with beads could be used for
RV-PCR analysis, as was done in this case. Although not tested in this effort, it may be possible
for the beads to be reused for subsequent rounds of IMS for the same sample by transferring the
0.5 mL bead solution to additional 60-mL aliquots and repeating the capture process. Although
there would be some bead loss for each round of IMS, using the same beads for multiple aliquots
of the same sample could lead to greater cell concentration factor.
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\h
Water sample
(up to 60 mL)
and 50 |jL
Y. pestis
Ab-coated beads
Figure 4 Pathatrix Iinmunomagnetic Separation system (Life Technologies, Inc.). The
system has the ability to concentrate cells up to 600-fold (60 mL down to 0.1 mL). Five samples
can be processed in 15 min during which the sample is passed across the magnet with antibody
(Ab)-coated beads about 400 times.
For the IMS experiment, cells were grown overnight in YPEB at 28°C with shaking at 180 rpm.
The OD600 was measured and the cells were washed with IX PBS. The cell suspension was
adjusted to approximately 10, 100, and 1000 cells per mL (based on dilutions of suspensions at
OD600 ~0.1). Triplicate three mL suspensions for each cell level were used for IMS. To each 3-ml.
sample, 30 mL IX PBS and 50 (uL antibody-coated beads were added, and the sample was
processed by the Pathatrix instrument (Figure 4). A negative control without Y. pestis cells was
also included. A flow chart for sample processing using IMS is shown in Figure 5. The Pathatrix
instrument processes five samples concurrently within 15 min, excluding sample handling, which
can add 10 min per set of five samples. Therefore, the overall time for RV-PCR analysis would
increase from approximately 36 to 40 hr for 48 samples by including IMS.
Direction of Flow
"V
a, * • •
JkAL
Capture phase Magnet
Antibody coated beads
Magnet for
bead capture
Bead collection
tube
Final volume
0.1 mL
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IMS-Treated Cells Control Cells
PCR analysis
PCR analysis
Take T24 aliquot (500 fiL)
20 jiL for
dilution/pla ting
Take T0 aliquot (500 juL)
Perform IMS
(Pathatrix system)
Incubate at 28°C
for 24 hr (180 rpm)
80 jliL for RV-
PCR; Add -3.3
rnL IX medium
3-mL for RV-PCR;
Add 300 juL 10X
medium
Recover 100 jiL
beads (~330-fold
concentration)
3-mL Y. pestis
suspension (Ave CFU/
mL ~3, 30, or 300)
3-mL Y. pestis
suspension (Ave CFU/
mL ~3, 30, or 300)
Add 30 mL PBS and
50 jiL Y. pestis-
specific Ab-coated
beads
Figure 5 Flow chart for IMS-treated and control Y. pestis cell suspensions.
Traditional culture analysis was used to determine recovery percentage relative to the inoculum
level from a portion of the recovered beads (20 [xL of total 100 [xL bead suspension), including
both direct plating (without dilution) of the resulting bead suspension and/or filter-funnel plating
(of the solution remaining after bead capture). The remaining bead solution (80 |iL) was added to
a 48-well plate with 3.3 mL IX YPEB. After mixing by pipettor, a To aliquot (500 |iL) was
removed and processed for DNA extraction as described in Section 2.8. Control cell suspensions
(3 mL for each cell level) were added to the 48-well plate and 300 |iL 10X YPEB was added,
mixed by pipetting up and down, and a To aliquot was removed for DNA extraction and PCR
analysis. The plate was incubated at 28°C for 24 hr with shaking at 180 rpm after which a 500 |iL
aliquot was removed for DNA extraction and PCR analysis.
2.15 Modified Filtration for Concentration of Y. pestis Cells
Modified filtration (MF) methods were investigated to prevent desiccation of cells on standard
membranes during vacuum filtration. In this task, different compounds were added to filter devices
(Whatman® Filtration Autovials™, GE Healthcare, Cat. No. AV125NPUPSU) to retain moisture
on the filter membrane to maintain cell viability during filtration. Autovials were qualified by
comparing their performance to that of the previously used Whatman® Autocups™ (Cat. No. 1602
- 0475), since the latter was discontinued; similar RV-PCR LOD results were obtained for the two
15
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different devices and the protocol was modified slightly to accommodate the smaller volume of
the Autovials (12.5 mL compared with 20 mL for the Autocups). In addition, vacuum manifolds
modified to accommodate the larger diameter Autovials were used.
Materials used to test MF included diatomaceous earth (DE) and different types of superabsorbent
polymers (SAPs). Due to its high silica content, DE has been used for nutrient and moisture
retention. SAPs have been used for similar and related applications including biosolids dewatering,
fuel filtering to remove water, diaper manufacturing, and as soil additives for moisture retention.
These materials are typically polymers of polyacrylic acid or co-polymers of poly(isobutylene)
and poly(maleic acid), which have different properties (absorption characteristics) dependent on
polymer chemistry and cross-linking. SAPs used in the testing included H-200, H-300, H-400, and
H-500 from JRM Chemical, Inc. (Cleveland, OH) with smaller numbers representing smaller
particle sizes) and 100% polyacrylamide "Water Storing Crystals" (Miracle-Gro™ abbreviated
MG; ScottsMiracle-Gro, Marysville, OH). DE was food-grade material packaged as "Kleen-N-
Fresh", which was obtained from Garden Fresh® (Pleasant Hill, CA). 0.1 to 0.2 gram (g) amounts
of SAP and/or DE materials were used per Autovial. The MF approach for cell concentration used
in this study is shown in Figure 6.
16
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Wash with PBS
Wash with
5-mL PBS
PCR analysis
PCR analysis
Cap Autovial
top;Incubate at
28°C for 24 hr
Take T0 aliquot
for DNA
extraction
Take T24 aliquot
for DNA
extraction
SAPs and/or DE
loaded into
Autovials
Cap bottom of
Autovial; Add
3-mLYPEB
medium
Add 5-mL cell
suspension;
complete
vacuum
filtration
Figure 6 Flow chart for sample processing using the modified filtration approach followed
by RV-PCR analysis for Y. pestis cells. Vacuum filtration of the Autovials was stopped as soon
as filtration was complete in a given vial to enable direct comparison between treatments for
biocompatibility and growth without assessing which material retained more moisture. The vials
with and without materials added were washed with 10 mL PBS before addition of cells.
17
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3. Quality Assurance and Quality Control
3.1 Laboratory Inspections
Monthly laboratory inspections were conducted by the project principal investigator (PI) to comply
with DOE 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 select agents (in addition, 25% inventory conducted quarterly)
• reviewing personnel training
3.2 Calibration
The Applied BioSystems™ Inc. (ABI) 7500 Fast PCR instrument was calibrated and underwent
preventative maintenance conducted annually. 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 PI.
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 pellets (for DNA processing) and DNA 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
Plating of the initial cell suspensions (or inoculum) and one or more negative samples (samples
spiked with phosphate-buffered saline, PBS) to test for cross-contamination were conducted for
each experiment.
3.5 Real-time PCR Analysis
During the experiment, Y pestis C092 extracted DNA 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. DNA standards were prepared from Y pestis C092
cells as described in the Materials and Methods section.
18
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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 cell suspension dilution
and processed at the same time following the same laboratory processes. Results are presented as
average Ct values (for the RV-PCR method) or average colony-forming units (CFU; for the spread
plate 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
cells 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 PCR analysis.
3.8 Data Quality Objectives/Data Quality Indicators
This research effort was to develop a qualitative, RV-PCR method of Y. pestis. 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 1 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 1 Ct value of the average). For individual replicates within an
experiment, the RV-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.
4. Results and Discussion
The following section presents results for the three project tasks and also provides some discussion
of results. In addition, particular details relevant to the given experiment are included such as cell
concentrations tested, broth used, and PCR assay employed. This allows the relevant information
to be in close proximity to the results to better understand the relationship between the experiment
variables and the data. The relevant Materials and Methods sections provide general information
whereas the paragraph(s) before the results description in this section provide specific information.
19
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4.1 TASK 1: Incorporate DNA Extraction and Purification Steps into RV-PCR Protocol
for Y. pestis and Evaluate Protocol Parameters (Incubation Period, LOD)
4.1.1. Objectives
As stated, the objective of the first task was to shorten the Y pestis RV-PCR method incubation
period by incorporating a DNA extraction/purification procedure, which also enabled
concentration of the resulting DNA. During the initial method development for Y pestis (US EPA
Internal Report, 2010), crude DNA extracts were obtained from samples by heat lysis with no
subsequent DNA concentration or cleanup performed. In this task, a DNA extraction/purification
procedure was incorporated into the protocol, which used chemical lysis to break open cells and
release their DNA. Specifically, the objective was to maintain the 10-cell level detection limit (10-
99 cells per sample) while significantly shortening the incubation from 48 hr; in this case a 24-hr
incubation period was targeted since a shorter incubation would lead to a shorter time for results
and increased sample throughput. However, in addition to chemical lysis used by the DNA
extraction/purification protocol, heat lysis followed by DNA concentration/purification was
evaluated as a potentially more streamlined approach. Since Y. pestis is a Gram-negative vegetative
cell and more easily lysed than Gram-positive cells, it was thought that the processing time could
be shortened and heat lysis could more quickly release DNA than chemical lysis steps.
4.1.2. Overall Approach for Evaluating DNA Extraction and Purification Protocols for Y.
pestis Cells
The RV-PCR protocol for B. anthracis used a MagneSil® Blood Genomic, Max Yield System kit
(Promega Corp., Madison, WI) consisted of several buffers for (i) cell lysis and recovery of DNA
onto magnetic beads, (ii) washes to purify the DNA, and (iii) elution of the purified DNA. The
reagents were shown to lyse the Gram-positive B. anthracis vegetative cells and not the spores. In
this effort, these reagents were evaluated for Y pestis cell lysis and DNA
concentration/purification. Simpler protocols with fewer reagents and steps were tested for ability
to generate DNA with sufficient quantity and quality for subsequent analysis. In particular, one of
the lysis wash steps and one of the alcohol wash steps were omitted.
In this task, Y pestis C092 cells were used for determining yield of DNA (based on PCR response)
from different extraction/purification procedures. Cell concentrations ranged from 101 to 106 per
sample and were determined for each experiment by serial dilution and plating. Real-time PCR
analysis used Y pestis-specific assays including YC2 (chromosome), YpMTl (pMTl), and YpPl
(pPCPl), which previously were down-selected based on assay sensitivity, ability to detect
inclusivity panel strains, and ability to not react with exclusivity panel strains (US EPA Internal
Report, 2010).
4.1.3. Evaluation of Y. /jesft's-Specific Real-Time PCR Assays
As mentioned, three real-time PCR assays were down-selected in the previous effort (US EPA
Internal Report, 2010). In the current effort, the sensitivity of the assays was confirmed using 10-
fold dilutions of prepared genomic DNA stocks from Y pestis C092. Genomic DNA was prepared
and quantified as described in the Materials and Methods Section 2.9. PCR Ct data are shown
below for 7-log standards for two plasmid assays, YpPl (pPCPl plasmid) and YpMTl (pMTl
plasmid), and the chromosomal assay, YC2.
20
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Results shown in Table 2 were comparable to values previously reported (US EPA Internal Report,
2010) for YC2 and YpMTl; however, the PCR response curve for YpPl was shifted to about 1-
log higher Ct values. Data showed good assay performance even down to the 5-fg level. The
greater sensitivity of the YpPl assay compared to the other assays could be due in part to there
being multiple pPCPl plasmids per cell due to its small size (-10 kb). In general, this effort
confirmed the selection of the three assays that were down-selected previously (US EPA Internal
Report, 2010).
Table 2 Real-time PCR Results for Y. pestis C092 Genomic DNA Dilutions With Assays for
pPCPl and pMTl Plasmids and the Chromosome
Y. pestis DNA
(Pg)
Average* Ct (SD) by Assay
YpPl
(pPCPl)
YC2
(chromosome)
YpMTl
(pMTl)
5000
16.9(0.3)
17.8 (0.2)
18.8 (0.6)
500
20.9 (0.4)
21.2 (0.2)
22.2 (0.1)
50
24.8 (0.3)
24.7 (0.2)
25.6(0.3)
5
29.4 (0.6)
28.4 (0.2)
29.6 (0.2)
0.5
33.7 (0.6)
32.0(0.2)
33.2 (0.5)
0.05
38.2 (0.6)
35.9(0.4)
37.2 (0.4)
0.005
41.6 (0.4)
38.0(1.2)
40.3 (0.4)
* Average and standard deviation (SD) based on four replicates.
4.1.4. Comparison of Cell Number Estimated from PCR Results and Viable Cell Counts -
Evaluation of Modified Chemical Lysis (Promega MagneSil) Protocol
In this effort, viable cell counts were compared with cell number estimates based on real-time PCR
results in order to estimate the efficiency of cell lysis and DNA concentration. The project scope
did not include an extensive optimization of the DNA extraction protocol but rather included a
smaller effort to assess DNA yield and quality (assessed together as PCR performance) when using
fewer wash steps. In this case, one fewer lysis wash step and one fewer alcohol wash step were
used compared to the protocol for DNA extraction of B. cmthracis cells (US EPA, 2012).
DNA target concentrations were estimated from the resulting Ct, using the standard curve and
assuming one target copy per cell and correcting for dilution (as described in Section 2.13). The
one copy per cell estimate was likely valid since the chromosomal assay YC2 was used. It was
also assumed that only live cells contributed DNA and that the dead cell population was negligible.
These assumptions seemed valid since 24 hr (T24) was not an excessively long incubation period
which would include entry into stationary phase for Y. pestis, this was supported by the measured
CFU/mL at T24 that ranged from about 4 x 105 to 3 x io7. The log difference between cell numbers
estimated from PCR analysis and those from culture varied from -0.2 to 0.7 with positive values
showing higher CFU based on PCR estimates and negative values showing higher CFU for culture
(Table 3). The average log difference for samples starting with 10, 100, or 1000 cells and using 24
21
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hr incubation was 0.4 ± 0.3 showing that results from PCR gave significantly higher estimates than
CFU values measured from plating. Many factors can affect the estimated CFU/mL for both
plating and PCR analysis (e.g., pipetting variability, variation in target copy number per cell) and
PCR does not generate absolute cell counts, but in general the results suggested that good DNA
yields were obtained using the modified DNA extraction protocol for Y. pestis cells.
Table 3 Comparison of Estimated CFU/mL from Real-Time PCR Analysis* (YC2 Assay)
With CFU/mL from Culture Analysis (With IX YPEB Prepared From 10X)
Inoculum
Log CFU/mL
from Culture
Sample
Replicate
Average Log
CFU/mL from
Culture at T24
Estimated
Average Log
CFU/mL from
PCRatT24**
Log Difference
(PCR -
Culture)
1
5.9
6.6
0.6
1.2
2
5.6
6.2
0.6
3
5.8
6.2
0.4
Ave (SD)
5.8 (0.2)
6.3 (0.2)
0.5 (0.1)
1
6.8
6.8
0.0
2.2
2
6.9
7.5
0.6
3
6.8
7.5
0.7
Ave (SD)
6.8 (0.1)
7.3 (0.4)
0.5 (0.4)
1
7.5
8.0
0.5
3.2
2
7.4
8.0
0.6
3
7.5
7.3
-0.2
Ave (SD)
7.5 (0.1)
7.8 (0.4)
0.3 (0.4)
Overall Ave
(SD)
0.4 (0.3)
* DNA extracts were obtained from T24 aliquots for RV-PCR analysis.
** Log CFU/mL was estimated assuming one target copy per cell, a genome size of 4.83 Mb (5.21 fg/target), using
the PCR Ct value at T24 corrected for dilution. A T24 aliquot of 500 |iL was used. Each sample replicate was
analyzed in triplicate by culture and PCR analyses.
CFU, colony forming units; SD, standard deviation.
22
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4.1.5. Comparison of PCR Results with Universal Reagents/Standard Cycling and Fast
Reagents/Fast Cycling Conditions
In addition to the data shown in Table 2 generated with Universal Master Mix and Standard cycling
(Universal/Standard) conditions, the same DNA standard concentrations were run with Fast
Master Mix and Fast cycling (Fast/Fast) conditions. The PCR cycling conditions for both Fast and
Standard Modes are shown in Table 4. Aside from different Master Mix used for the different PCR
conditions, the same concentration of primers and probe and the same concentration of DNA
standards were used. The average Ct differences across 7-log Y pestis genomic DNA
concentrations for the two PCR conditions were 4.5 ±1.3 and 1.7 ± 0.2 for YpPl and YpMTl,
respectively (Table 5); the YC2 assay did not show differences (Table 6).
Table 4 Thermal Cycling Parameters for the Different Real-Time PCR Configurations
Cycling Type
(7500 Fast
System)
Thermal-Cycling Profi
e
Overall
PCR Run
Time
(hh:mm)**
Parameter
UNG
Incubation f
Polymerase
Activation J
PCR (45 cycles)
Hold
Hold
Denature
Anneal/
Extend*
Temp. (°C)**
50
95
95
60
Standard Mode
(Universal
Master Mix)
Time (mm:ss)
02:00
10:00
00:05
00:20
00:55
Fast
Mode (Fast
Master Mix)
Time (mm:ss)
02:00
00:20
00:03
00:20
00:45
f Required for optimal UNG activity.
{ Required to activate the DNA polymerase.
* Based on a single-channel (FAM) measured; if all four channels were measured the Anneal/Extend period would
need to be extended to 01:00 for Standard Mode and 00:30 for Fast Mode.
** hh = hour; mm = minutes, and ss = seconds (in double digit format).
Table 5 Real-time PCR Results for the Plasmid Assays YpPl (pPCPl) and YpMTl (pMTl)
Using Fast/Fast and Universal/Standard Conditions With Y. pestis DNA Standards
F. pestis
DNA (pg)
YpPl Assay Ave Ct (SD)
Ct
Difference
YpMTl Assay Ave Ct
(SD)
Ct
Difference
Fast/Fast
Universal/
Fast/Fast
Universal/
Standard
Standard
5000
14.2 (0.1)
16.9 (0.3)
2.7
17.2 (0.1)
18.8 (0.6)
1.6
500
17.7(0.2)
20.9 (0.4)
3.2
20.8 (0.1)
22.2 (0.1)
1.4
50
21.2 (0.3)
24.8 (0.3)
3.6
24.1 (0.1)
25.6 (0.3)
1.5
5
24.4 (0.1)
29.4 (0.6)
5.0
27.7(0.1)
29.6 (0.2)
1.9
0.5
28.4 (0.1)
33.7 (0.6)
5.3
31.7(0.3)
33.2 (0.5)
1.5
0.05
31.8 (0.1)
38.2 (0.6)
6.4
35.4 (0.1)
37.2 (0.4)
1.8
0.005
36.3 (0.8)
41.6 (0.4)
5.3
38.2 (0.3)
40.3 (0.4)
2.1
Ave (SD)
4.5 (1.3)
Ave (SD)
1.7 (0.2)
* Average (Ave) and standard deviation (SD) based on 3-4 replicates, pg = picogram.
23
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Table 6 Real-time PCR Results for the YC2 (Chromosomal) Assay Using Fast/Fast and
Universal/Standard Conditions With Y. pestis DNA Standards
YC2
F. pestis
DNA (pg)
Assay Ave Ct (SD)
Ct
Difference
Fast/Fast
Universal/
Standard
5000
18.2 (0.1)
17.8 (0.2)
-0.4
500
21.6(0.1)
21.2 (0.2)
-0.4
50
24.9(0.1)
24.7 (0.2)
-0.2
5
28.5 (0.1)
28.4 (0.2)
-0.1
0.5
32.3 (0.2)
32.0 (0.2)
-0.3
0.05
36.2 (0.8)
35.9 (0.4)
-0.3
0.005
38.3 (0.1)
38.0 (1.2)
-0.3
Ave (SD)
-0.3 (0.1)
* Average (Ave) and standard deviation (SD) based on 3-4 replicates, pg = picogram
The larger differences especially for the YpPl assay which targets a ~10-kb plasmid could be due
to plasmid supercoiling and the possibility that the Fast reagents and Fast cycling program
amplified supercoiled DNA more efficiently. Since results were similar between the two
modes/conditions for the YC2 assay, the Universal/Standard condition was used for the subsequent
experiments with the YC2 assay; however, Fast reagents and Fast cycling profiles could be
considered for improved sensitivity using the plasmid assays.
4.1.6. Evaluation of Heat Lysis Vs. Chemical Lysis for Y. pestis Cells
Since heat lysis could simplify the DNA extraction/purification procedure in the RV-PCR protocol
(based currently on chemical lysis with Promega reagents), both lysis methods were tested in
parallel with Y. pestis C092 cells. After the initial lysis step, the purification steps were the same
between both procedures (Figure 7). Lysis buffer was added to heat lysates only to ensure proper
chemistry for DNA binding and the samples were not incubated in this buffer. The goal was to
determine if the modified protocol provided the same DNA yield and quality (as determined by
real-time PCR analysis) as the original protocol. Cells were from overnight cultures which were
diluted to OD600 ~0.1. One-mL aliquots were processed to recover DNA using the standard DNA
extraction/purification protocol, after initially concentrating the cell suspension to 200 [xL by
centrifugation (as described in Materials and Methods Section 2.8). All three assays were used for
PCR analysis, YC2, YpPl and YpMTl.
24
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Protocol Stei) Chemical Lysis Heat Lysis
Aliquot Sample
Harvest Cells
Remove Supernatant
J 'ortex Cell Pellet
Lysis
Alcohol Wash
EtOU Wash
Dry Beads
2X 500 jiL
IX 500 nL
2X 320 jiL
Centrifuge cells
Remove 800 jiL
Centrifuge cells
Remove 800 jiL
Suspend in 200 jiL
Suspend in 200 jiL
1-inL Cell suspension
1-inL Cell suspension
300 (iL added:
-150-200 fiL
recovered
Add 800 (iL
Lysis Buffer
(0 mill) -DNA
Capture
IX 800 |iL Lysis
Buffei (5 mill) -
DNA Capture
IX 320 fiL Lysis
Buffei' Wash
Figure 7 Outline of Protocol Steps for Chemical Lysis (Promega MagneSil kit) and Heat
Lysis Procedures for DNA Extraction and Purification. For the heat lysis procedure, 800 [xL
Lysis Buffer was added after heat treatment to create proper conditions for released DNA to bind
to MagneSil magnetic beads. The samples were put immediately on a magnetic rack and the
liquid was removed to limit chemical lysis activity.
Results showed the overall process times were similar for the different lysis treatments;
approximately 3 hr for 24 samples processed manually. This is in part due to the fact that the same
steps were used after lysis. RV-PCR results from triplicate samples and triplicate PCR analyses
per sample replicate showed that DNA extracts from chemical lysis treatment produced
significantly lower average Ct values (3-4 units lower) for the first replicate experiment, ranging
from 16.6-19.8 compared to 20.5-23.0 for heat lysis for all replicates and assays (Table 7) with
p-values ranging from 2.2 x 10"5 to 7.5 x 10"7 (paired, two-tailed T-test). The data suggested that
chemical lysis was more effective for reproducibly generating good quality, amplifiable genomic
DNA. Diluted extracts (10-fold) showed the same trends although the ranges overlapped with Ct
values of 20.6-23.2 and 22.6-24.3, for chemical and heat lysis treatments, respectively. The data
suggested some level of PCR inhibition for heat lysates that was not observed for extracts from
chemical lysis.
25
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Table 7 Real-time PCR Results for DNA Extracted from Y. pestis Cells by Heat or
Chemical Lysis (followed by Promega Kit Purification) and Analyzed by Chromosomal
(YC2) and Plasmid Assays (YpPl and YpMTl) - First Replicate Experiment
Sample
Replicate-
PCR
Replicate
Ct* by Y. pestis Assay
DNA
YpPl
YC2
YpMTl
Extraction
Method
Undiluted
10-Fold
Dilution
Undiluted
10-Fold
Dilution
Undiluted
10-Fold
Dilution
1-1
22.2
23.3
21.8
23.8
23.5
25.3
1-2
21.7
23.4
21.3
23.9
23.9
25.3
1-3
21.4
24.0
21.1
24.1
24.1
25.7
Ave (SD)
21.8 (0.4)
23.6 (0.4)
21.4 (0.4)
23.9 (0.1)
23.9 (0.3)
25.4 (0.2)
2-1
20.6
21.7
20.2
22.5
22.6
23.7
2-2
20.3
22.2
19.8
22.3
22.3
23.8
Heat Lysis
2-3
19.8
22.4
19.8
22.9
22.6
23.9
Ave (SD)
20.2 (0.4)
22.1 (0.3)
19.9 (0.2)
22.6 (0.3)
22.5 (0.2)
23.8 (0.1)
3-1
20.3
21.7
20.5
22.6
22.5
23.5
3-2
19.8
22.1
19.9
22.8
22.1
23.8
3-3
20.3
22.4
20.2
22.7
22.8
23.9
Ave (SD)
20.2 (0.3)
22.1 (0.3)
20.2 (0.3)
22.7 (0.1)
22.5 (0.4)
23.7 (0.2)
Overall
Ave (SD)
20.7 (0.8)
22.6 (0.8)
20.5 (0.7)
23.1 (0.7)
23.0 (0.7)
24.3 (0.8)
1-1
17.0
20.5
18.5
22.1
20.3
23.4
1-2
16.7
20.6
18.6
22.2
20.1
23.5
1-3
16.8
21.1
18.7
22.6
19.9
23.7
Ave (SD)
16.8 (0.1)
20.7 (0.3)
18.6 (0.1)
22.3 (0.3)
20.1 (0.2)
23.5 (0.2)
2-1
16.7
20.4
18.7
21.9
20.2
22.9
Promega
2-2
16.5
20.6
18.4
21.9
19.6
23.1
Kit
2-3
16.5
20.7
18.2
22.1
19.8
23.7
(Chemical
Lysis)
Ave (SD)
16.6 (0.1)
20.6 (0.2)
18.4 (0.2)
22.0 (0.1)
19.9 (0.3)
23.2 (0.4)
3-1
16.4
20.2
18.2
21.7
19.5
22.9
3-2
16.6
20.5
18.2
21.7
19.2
22.8
3-3
16.5
20.7
18.2
22.0
19.4
23.3
Ave (SD)
16.5 (0.1)
20.5 (0.2)
18.2 (0.0)
21.8 (0.2)
19.4 (0.1)
23.0 (0.2)
Overall
Ave (SD)
16.6 (0.2)
20.6 (0.2)
18.4 (0.2)
22.0 (0.3)
19.8 (0.4)
23.2 (0.3)
* Ct = cycle threshold; SD = standard deviation. Average (Ave) and SD values are based on triplicate samples.
A replicate experiment was conducted using the same conditions as described above. PCR results
in terms of averages and standard deviations from three biological replicates and triplicate PCR
analyses are shown in Table 8. Unlike the first replicate experiment, there were much smaller
differences (0.1-0.8) between heat lysis and chemical lysis treatments with average Ct values for
undiluted extracts ranging from 16.4-19.4 and 16.3-18.8 across assays, respectively. As
mentioned, the experiments were conducted in the same manner although the estimated cell
number differed slightly; OD600 values were 0.11 and 0.18 for the first and second experiments,
26
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respectively, which corresponds to about 1.6-fold difference in estimated cell numbers. In general
the Ct values for the first replicate experiment were higher since fewer cells were used for this
experiment. Although there were differences in results, the data suggested that chemical lysis
provided more consistent, lower Ct values (more DNA recovery) therefore this extraction method
was used in the RV-PCR protocol for Y pestis analysis.
Table 8 Real-time PCR Results for DNA Extracted from Y. pestis Cells by Heat or
Chemical Lysis (followed by Promega Kit Purification) and Analyzed by Chromosomal
(YC2) and Plasmid Assays (YpPl and YpMTl) - Second Replicate Experiment
Sample
Replicate
-PCR
Replicate
Ct* by Y. pestis Assay
DNA
YpPl
YC2
YpMTl
Extraction
Method
Undiluted
10-Fold
Dilution
Undiluted
10-Fold
Dilution
Undiluted
10-Fold
Dilution
1-1
18.3
19.9
17.4
18.1
20.9
21.3
1-2
17.9
19.9
17.3
17.9
20.2
20.9
1-3
17.6
19.7
17.0
18.4
19.6
21.2
Ave (SD)
17.9 (0.4)
19.8 (0.1)
17.2 (0.2)
18.2 (0.3)
20.3 (0.7)
21.1 (0.2)
2-1
17.2
20.0
17.0
18.4
19.8
20.9
2-2
17.6
20.1
16.4
18.5
19.2
20.8
Heat Lysis
2-3
17.5
20.1
15.6
18.7
18.6
21.3
Ave (SD)
17.4 (0.2)
20.1 (0.1)
16.3 (0.7)
18.5 (0.1)
19.2 (0.6)
21.0 (0.3)
3-1
18.0
19.8
15.9
18.2
19.3
20.7
3-2
17.5
19.7
15.7
18.3
18.9
20.6
3-3
16.8
19.9
15.4
18.3
17.9
20.8
Ave (SD)
17.4 (0.6)
19.8 (0.1)
15.7 (0.2)
18.3 (0.1)
18.7 (0.7)
20.7 (0.1)
Overall
Ave (SD)
17.6 (0.5)
19.9 (0.2)
16.4 (0.8)
18.3 (0.2)
19.4 (0.9)
20.9 (0.2)
1-1
17.3
19.6
16.4
17.9
19.2
20.1
1-2
16.6
19.7
15.7
18.0
18.0
20.3
1-3
16.5
19.6
15.4
18.1
17.7
20.1
Ave (SD)
16.8 (0.4)
19.6 (0.1)
15.8 (0.5)
18.0 (0.1)
18.3 (0.8)
20.2 (0.1)
2-1
17.0
19.8
17.1
18.7
20.3
20.6
Promega
2-2
16.9
19.7
16.3
18.2
18.4
20.7
Kit
2-3
16.7
19.7
16.3
18.6
18.3
20.8
(Chemical
Lysis)
Ave (SD)
16.9 (0.2)
19.7 (0.0)
16.6 (0.4)
18.5 (0.3)
19.0 (1.1)
20.7 (0.1)
3-1
17.3
20.1
16.9
18.7
19.2
21.0
3-2
17.0
20.1
16.5
18.7
20.0
20.9
3-3
16.8
20.2
15.7
18.7
18.1
21.1
Ave (SD)
17.1 (0.3)
20.1 (0.0)
16.4 (0.6)
18.7 (0.0)
19.1 (1.0)
21.0 (0.1)
Overall
Ave (SD)
16.9 (0.2)
19.8 (0.2)
16.3 (0.6)
18.4 (0.3)
18.8 (0.9)
20.6 (0.4)
* Ct = cycle threshold; SD = standard deviation. Average (Ave) and SD values are based on triplicate samples.
27
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With the RV-PCR method using chemical lysis, the overall sample processing and analysis time
for 48 samples and controls (3 mL volume) would be -36 hr including: 2-3 hr for sample receipt
and set up in 48-well plates and taking the To aliquots; 24 hr for incubation (during this time, To
aliquots are processed for DNA extraction); 3-4 hr for taking the T24 aliquots and processing the
first set of 24 samples/controls for DNA extraction; ~3 hr for DNA extraction of the second set of
24 samples/controls; and ~3 hr for PCR setup and analysis (To and T24 DNA extracts for all 48
samples/controls). In addition, multi-channel pipettors or automated platforms may be used with
RV-PCR to further enhance throughput and shorten the time to results.
4.2 TASK 2: Further development and optimization of sample processing protocols for Y.
pestis cell recovery and growth
4.2.1. Objectives
The objectives of this task were to optimize the recovery efficiency of Y pestis cells from water
samples and enhance subsequent growth kinetics in the RV-PCR format. Culture conditions in 48-
well plates were modified to enhance growth kinetics with the goal of shortening the method
incubation period from 48 hr to 24 hr. This represented a focused effort in which different liquid
growth media were evaluated such as YPEB (Doran et al., 2013) recommended by Dr. Darcy
Hanes (FDA) (personal communication), and BHI broth as used in a previous effort (US EPA
Internal Report, 2010) and by Gilbert et al. (2014). Aliquots were removed overtime with care to
not significantly deplete the culture volume and affect the growth rate; in this regard separate 48-
well plates were set up for different incubation periods so that only one aliquot each were removed
for the To and Tftime points. Serial dilution and plating was also conducted at the same end points
to assess cell growth. The overall goal was to leverage the results from optimization of cell
recovery and growth conditions (from this task) and optimized methods for recovery of purified
DNA from cells from Task 1, such that an improved RV-PCR protocol could be evaluated in Task
3.
4.2.2. Approaches Used for Y. pestis C092 Growth Optimization
Initial culture was performed using BHI agar plates; however, poor growth was observed such that
TBA (base without blood; Becton Dickinson Difco™, Cat. No. 223220) was substituted. TBA was
more consistent often showing >2-fold higher plate counts from the same cell suspension and
sometimes providing data where no counts were obtained for BHI plates. Results reported below
are based on TBA plate counts corrected for dilution. For propagation of broth cultures in the 48-
well plate format, initial experiments tested BHI broth at IX concentration, followed by testing of
broth prepared with nine parts PBS (for cell suspensions) or distilled deionized water (for only
broth reconstitution) and one part 10X BHI broth. Subsequent experiments tested YPEB in both
the IX and reconstituted formats as for BHI broth.
4.2.3. Evaluation of Y. pestis Growth in IX BHI Broth in 48-Well Plate Format
Growth experiments were initiated with -20°C glycerol stock of Y. pestis C092 and used 5 mL
overnight cultures as described in the Materials and Methods section. Three successive overnight
cultures in BHI broth were prepared by diluting to an OD600 -0.1 using a portable UV
spectrophotometer) and propagated overnight (18-26 hr) at 28°C with orbital shaking at 180 rpm.
Initially, successive overnight cultures were propagated based on previous reports of improved
28
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growth in BHI broth using this approach; however, later experiments used only one overnight
culture as described. After overnight incubation, the OD600 values were -1.8 to > 2 (i.e., the
maximum reading on the UV spectrophotometer). To set-up the 48-well plate, the culture was
again diluted to OD600 value of- 0.1 which corresponded to - 1 x 107 CFU/mL (actual counts from
plating were -6-7 x 106 CFU/mL). Subsequent 10-fold dilutions were performed in BHI broth to
produce cell suspensions down to - 1 x 101 CFU/mL; 1 x 102 — 1 x 104 cell concentrations were
also included. Each well contained three mL of culture at the appropriate dilution and triplicates
were included per cell level. The plate also contained negative controls with broth although without
cells. Plates were covered with adhesive AeraSeal (Molecular Devices, Sunnyvale, CA) and
incubated at 28°C (with shaking at 180 rpm) for 24 and 40 hr. Separate plates were prepared in the
same manner and used for each time point.
The growth curves from a 48-well plate experiment (based on TBA plate counts) are shown in
Table 9. Results showed about 1-log increase over 24 hr for 6 x 101 and 6 x 102, and a > 3-log
increase for 6 x 103. The inoculum with -6 CFU/mL did not increase over time. These data were
similar to results obtained previously using Y pestis with BHI broth (US EPA Internal Report,
2010); the study showed a lower limit for Y pestis growth in 48-well plates, between 10 and 100
CFU - at least for observable growth in 24 to 48 hr.
Table 9 Growth of Y. pestis Cells in 48-Well Plates (3 mL IX BHI)* for ~6 - 6 x 103 Colony
Forming Units (CFU)/mL Starting Y. pestis Cell Concentrations
Time
Point
Average (SD) Measured CFU/mL for Different Starting Cell
Concentrations (CFU/mL)**
6
60
600
6000
0
5.9 (1.6) x 10°
5.9 (1.6) x 101
5.9(1.6) x 102
5.9(1.6) x 103
24
3.0 (1.7) x 101
7.7 (8.3) x 102
1.8 (0.4) x 103
4.1 (0.7) x 107
40
1.5 (0.7) x 101
3.8 (3.0) x 105
1.8 (0.1) x 107
1.4(0.1) x 10s
Log
Increase
0.7
1.1
0.5
3.8
Tf
H
1
O
H
Log
Increase
0.4
3.8
4.6
4.4
(T0-T40)
* Cells were prepared by three sequential overnight cultures, harvested by centrifugation, suspended in IX BHI,
transferred to 48-well plates (3 inL), and incubated at 28°C (180 rpm) for 24 or 40 hr.
** Results are averages and standard deviations (SD) from triplicate samples with one replicate plate count per
sample. Data represent the average and SD from inoculum reference plating, corrected for dilution.
At each time point, a 1-mL aliquot from each sample (as well as two negative controls without
cells) was extracted for DNA using the Promega Magnesil reagents. The protocol for Y pestis cells
was based on that for B. anthracis (US EPA, 2012) and included the following: (1) two lysis buffer
steps; (2) two salt wash buffer steps; (3) two alcohol wash buffer steps; (4) one 70% ethanol wash
buffer step; (5) bead drying; and (6) DNA elution. PCR was performed on undiluted DNA extracts
using the YC2 assay with 45 amplification cycles. The resulting Ct data were used to estimate Y
pestis cells based on Y pestis genomic DNA standard curves assuming one target DNA copy per
cell and assuming the size of a Y pestis genome is 4.83 Mb (see Materials and Methods Section
2.13).
29
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The data showed that good DNA yields were obtained from the Promega kit for the 48-well
cultures with estimated DNA copies greater on average than those measured from culture analysis.
For all assays, the difference between estimated cell numbers from PCR analysis and measured
cell numbers from culture analysis was 0.4 ± 0.6 log higher for all assays and for both time points
(0.5 ± 0.5 for the YC2 chromosomal assay) (see Section 2.13 for calculations). The estimates have
inherent error due to assumptions for converting Ct data to cell number.
4.2.4. Evaluation of Y. pestis Growth in IX BHI Broth (Prepared From 10X BHI)
A similar experiment was conducted (with multiple overnight cultures) where in this case Y. pestis
cells were harvested by centrifugation and washed with IX phosphate-buffered saline (PBS) prior
to setting up the cell suspensions in a 48-well plate. Cell suspensions were diluted to different
starting concentrations as described previously and prepared with 10X BHI broth to yield IX BHI.
This was done to match the conditions that would be used for RV-PCR analysis of water samples,
which would be mixed with 10X BHI to yield IX concentration. The overnight cultures used BHI
prepared from 10X BHI as well.
Results are shown in Table 10 for plates incubated at 28°C (with shaking at 180 rpm) for 24 and
40 hours, again with separate plates used for each time point. The data showed better growth after
24 hr in this case for -8-800 CFU/mL, and comparable growth for -8,000 CFU/mL. Unlike the
earlier experiment, significant growth was observed for the 6 CFU/mL level. The growth after 40
hr was similar for all but the -6 CFU/mL level, which as mentioned did not show growth over
time in the previous experiment. It should be noted that plate counts for initial cell concentrations
-6 and 60 CFU/mL at 40 hr were actually above the counted CFU since the plate dilution was
missed and plates were too numerous to count (TNTC); the data points are shown at greater than
the plate count limit corrected for dilution. Overall, the data suggested that use of reconstituted
broth to generate IX BHI would work well for the protocol with actual water samples.
Table 10 Growth of Y. pestis Cells in 48-Well Plates (3 mL BHI Prepared Using 10X BHI)*
Time Point
Average (SD) Measured CFU/mL for Different Starting Cell Concentrations
(CFU/mL)**
8.4
84
840
8400
0
8.4 (0.6) x 10°
8.4 (0.6) x 101
8.4 (0.6) x 102
8.4(0.6) x 103
24
4.4(3.1) x 102
4.2 x 104t
3.9 x 105t
6.2(0.8) x 106
40
> 3 x 104***
> 3 x 105 ***
1.7(0.2) x 107
4.1 (1.1) x 107
Log Increase
(To-T24)
1.7
2.7
2.7
2.9
Log Increase
(T0-T40)
>3.6
>3.6
4.3
3.7
* Cells were prepared by three sequential overnight cultures, harvested by centrifugation suspended in IX PB S, mixed
with 10X BHI in 48-well plates to yield IX BHI (3 mL), and incubated at 28°C (180 rpm) for 24 or 40 hr.
** Data points show the average and standard deviation (SD) from triplicate analyses for inoculum reference plating
(0 time point) and after 24 and 40 hr incubation, corrected for dilution.
*** Values for 6 and 60 CFU/mL at T4o were greater than the values shown due to incorrect dilutions plated.
' Data are from single replicates.
30
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As for the experiment using IX BHI (Section 4.2.3), a 1-mL aliquot was taken at T24 or T40 from
each sample and negative control for DNA extraction and purification; one aliquot was taken per
each well since two separate 48-well plates were used for each incubation period, T24 or T40.
Likewise, good DNA yields were obtained such that the estimated CFU/mL from PCR data was
on average within 2-fold of the expected value based on plate counts (with 0.0 ± 0.2 log
representing the difference between PCR and culture analysis for both time points across all assays,
and a 0.1 ± 0.2 log difference for the YC2 assay) (see Section 2.13 for calculations). As mentioned,
the estimates for cell number from PCR data have inherent error due to assumptions for converting
Ct data to cell number and variability in pipetting and standard curve generation.
4.2.5. Evaluation of Y. pestis Growth in IX YPEB Compared to IX BHI Broth Prepared
From 10X BHI Broth
An experiment was conducted comparing Y pestis growth on IX BHI (prepared using 10X BHI)
and IX YPEB. In this case, a single overnight culture of Y pestis cells in either IX BHI or IX
YPEB was generated, harvested by centrifugation (15 min at 4,000 rpm at 4°C), washed in PBS
buffer, and diluted to -10 to ~104 CFU/mL in IX BHI broth (prepared using 10X BHI broth) or
IX YPEB in two separate 48-well plates. The actual average CFU/mL from plate counts at To were
-3.5 to -3.5 x 103 for BHI and -2.8 to -2.8 x 103 for YPEB. The total culture volume per well
was 3 mL. The 48-well plates were incubated at 28°C (with shaking at 180 rpm).
Results showed poor growth for BHI-grown cells (data not shown), demonstrating inconsistency
with previous experiments possibly due to use of a single overnight culture rather than three
overnight cultures. However, results from YPEB-grown cells (from a single overnight culture)
showed significant increases in cell density representing a 3.6 to 4-log increase over the 24-hr
period (Table 11). Based on inconsistent growth on BHI, Y pestis C092 cells were only
propagated on YPEB for the remainder of the experiments.
Table 11 Growth of Y. pestis Cells in 48-Well Plates (3 mL IX YPEB)*
Average (SD) Measured CFU/mL for Different Starting Cell Concentrations
Time Point
(CFU/mL)**
3
30
300
3000
0
2.8 (0.1) x 10°
2.8 (0.1) x 101
2.8 (0.1) x 102
2.8 (0.1) x 103
24
2.4(1.2) x 104
1.7(0.6) x 105
1.5 (0.1) x 106
8.5 (2.0) x 106
Log Increase
(T0-T24)
4.0
3.9
3.8
3.6
* Cells were prepared by one overnight culture, harvested by centrifugation, suspended in IX YPEB, added to 48-
well plates (3 mL), and incubated at 28°C (180 rpm) for 24 hr.
** Data points show the average and standard deviation (SD) from triplicate analyses for inoculum reference plating
(0 time point) and after 24 hr incubation corrected for dilution.
4.2.6. Growth of Y. pestis in 48-Well Plates and RV-PCR Analysis With Different To and Tf
Aliquot Volumes
An experiment was conducted with IX YPEB (prepared from 10X YPEB similar to that described
for preparation of IX BHI broth from 10X BHI broth) to evaluate the impact of different aliquot
volumes on Y pestis growth and ACt values at two different incubation periods, 12 and 24 hr. Y
31
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pestis cells were prepared as described above and used to inoculate YPEB. Y. pestis cells from a
single overnight culture were harvested, washed in PBS buffer, and diluted to -10 to 103 CFU/mL
(the actual CFU/mL were -14-1.4 x 103 from plating). The experiment used different 48-well
plates for each incubation period. Different aliquot volumes including 250 [xL and 500 [xL were
tested, with the same volume used for To and T12 or To and T24 (the total culture volume per well
was 3 mL). In addition, colony counts were obtained from these time points by serial dilution and
plating onto TBA plates and used to assess growth with IX YPEB prepared with 10X YPEB in
the 48-well format. Aliquots were centrifuged and the supernatant was removed to leave 200 [xL
for DNA extraction for both aliquot volumes to allow direct comparison of results. The Promega
MagneSil kit was used as described in the Materials and Methods Section 2.8 and resulting extracts
were analyzed undiluted using the YC2 assay with 45 amplification cycles.
Culture analysis showed -4.3-4.8 log increase over a 24-hr period and ~2-log increase during 12-
hr incubation. This experiment showed that even with removal of either 250 or 500 [xL at To
(including cells present in this aliquot), good growth conditions were observed. Results from PCR
analysis of the extracts are shown in Table 12 in terms of ACt for the two different incubation
periods. For each starting cell level, the 500 [xL extract gave higher average ACt values. For T12,
ACt ranged from 5.9-7.3 for the 250 [xL aliquots and 6.3-9.6 for T12 for the 500 [xL aliquots. For
T24, ACt ranged from 13.2-17.7 for the 250 [xL aliquots and 15.3-19.8 for the 500 [xL aliquots.
While it was expected that the 500 [xL aliquot volume would contain DNA from twice as many
cells as that for the 250 [xL aliquot, it also resulted in twice as many cells being removed at To such
that they could not contribute to cell propagation. Statistical analyses of culture and PCR results
for samples processed using 250 or 500 [xL volumes for both To and T24 did not show significant
differences for the different volumes (p-values ranged from 0.1 to 0.9); however, since average
ACt values for 500 [xL aliquots were greater than those for 250 [xL aliquots, 500 [xL aliquots were
used for subsequent RV-PCR experiments.
32
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Table 12 Effect of Time Point Aliquot Volume (250 and 500 jiL) on ACt for RV-PCR
Analysis: Y. pestis Cells in YPEB (Prepared Using 10X YPEB)*
Starting
CFU/mL
Aliquot
Volume
ACt** (YC2 Assay)
H
O
1
H
to
Tf
rt
H
1
®
H
Ave
SD
Ave
SD
103
250 (j,L
6.9
0.3
16.0
0.4
6.0
0.3
12.8
0.2
5.2
0.4
10.7
0.1
Ave
6.1
0.8
13.2
2.3
500 (j,L
6.2
0.2
14.8
0.1
7.0
0.1
15.0
0.3
5.8
0.3
16.0
0.2
Ave
6.3
0.6
15.3
0.6
102
250 (j,L
6.9
0.1
16.0
0.8
3.4
0.5
16.1
0.8
7.3
0.7
15.9
0.9
Ave
5.9
1.9
16.0
0.7
500 (j,L
7.1
0.4
17.3
0.6
7.6
1.4
15.8
0.5
5.9
0.8
17.3
0.7
Ave
6.9
1.1
16.8
0.9
101
250 (j,L
6.5
0.4
17.2
0.1
6.8
0.7
18.6
0.1
8.6
0.2
17.1
0.1
Ave
7.3
1.1
17.7
0.7
500 (j,L
10.5
0.0
20.3
0.1
9.4
0.2
20.2
0.1
OO
*00
0.3
19.0
0.1
Ave
9.6
0.8
19.8
0.6
* Cells were prepared from one overnight culture, harvested by centrifugation, suspended in IX PBS,
reconstituted with 10X YPEB in 48-well plates to yield IX YPEB (3 inL), and incubated at 28°C (180 rpm) for
24 hr.
** Average and standard deviation (SD) were based on triplicate samples.
Results also showed that good DNA yields were obtained corresponding to an average of 0.2 ± 0.3
log higher CFU/mL estimated from PCR results relative to those measured by plate counts
(corrected for dilution) from the same sample time point (see Section 2.13 for calculations).
Greater estimated CFU/mL from PCR analysis compared to culture analysis likely resulted from
assumptions in copy number calculation and variations in pipetting, PCR and culture efficiency.
It should also be reiterated that PCR data cannot be used to determine absolute cell counts.
Regardless, the data showed DNA extraction and purification procedures using the Promega
reagents were quite effective. Based on these results, IX YPEB (prepared from 10X) was used for
subsequent Y. pestis cultures and the 500 |iL aliquot volume was selected for RV-PCR analysis.
33
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4.2.7. Modified Filtration for Concentration of Y. pestis Cells from Larger Volume Water
Samples
With regard to concentration and recovery of Y pestis cells, the methods developed for B.
anthracis spores needed to be modified for vegetative cells to obtain improved limits of detection.
Unlike spores, vegetative cells are susceptible to killing by desiccation during filtration. The
feasibility of a modified filtration (MF) approach was evaluated for Y pestis cells from complex
samples to allow filtration and retain moisture for collection of viable Y pestis cells. Compatibility
with RV-PCR analysis was also investigated in a proof-of-principle MF test using SAPs with
different particle sizes and chemical compositions and DE in current filtration devices, like those
used for collection and concentration of B. anthracis spores. The SAP materials were inherently
inexpensive since many processes use them in large quantities. Filtration properties (i.e., speed),
moisture retention, and biocompatibility (i.e., cell viability maintenance and cell outgrowth) were
evaluated, with the initial down-selection based on filtration properties. Different SAP types and
amounts were tested with Whatman™ Autovials since the previously used Autocups were
discontinued by the vendor (GE Healthcare). Autovials use similar membrane materials
(polyethylene sulfone compared with nylon) and the same pore size, 0.45 micron; however, their
volume capacity is 12.5 mL, which is less than that for the Autocups, 20 mL, such that lower
volumes must be filtered at a time. Vacuum manifolds were modified to hold the wider Autovials
on a parallel Department of Homeland Security project. Different top and bottom caps were also
identified for the new filter vials in order to perform sample incubation following cell collection
and broth addition. A flow chart in Materials and Methods Section 2.15 shows the MF sample
processing method used upstream of RV-PCR analysis.
Prior to this study, it was not known whether the diatomite material could retain sufficient moisture
to maintain cell viability or whether the material was sufficiently biocompatible to allow cell
propagation. It should be noted that excessive vacuum durations were not used to fully test
desiccation of the SAPs or DE materials in this study; the assessment was simply a proof-of-
principle study to see how the materials functioned in the filter devices (Autovials) and whether
they were also biocompatible with Y pestis cells. In this regard, individual filter samples were
under vacuum until the liquid had completely filtered and then the vacuum was released, thereby
removing the variable of drying (or desiccation) time.
For the proof-of-principle study, DE, aqueous-based SAPs H-400, and H-500 (JRM Chemical,
Inc.), and "Water Storing Crystals" (Miracle-Gro, MG; another type of SAP) were used. Initial
studies focused on physical properties of wetting and filtration using different amounts of SAPs
and/or DE. Based on the initial findings, an RV-PCR experiment was initiated to evaluate different
amounts of DE, MG, and SAPs alone or in combination with other materials, as follows:
1) 0.1 gDE
2) 0.2 gDE
3) 0.1 gDE plus 0.1 g MG
4) O.lgMG
5) 0.1 gH-300 plus 0.1 g H-400
6) Control without SAPs, MG, or DE
34
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Y pestis cells were diluted from an overnight culture to 4 x 104 CFU in 5 mL PBS (determined
from reference plating onto TBA plates), and added to pre-washed SAPs and/or DE materials in
Autovials. After filtering the cell suspension, the vials were rinsed with 10 mL PBS. In each case,
vacuum filtration was stopped as soon as filtration was complete in a given Autovial. This allowed
direct comparison between treatments without assessing which material retained more moisture
for given vacuum conditions. The only treatment that did not slow the filtration speed relative to
the control (Autovial alone) was 0.1 g MG; other treatments added 1 min to > 5 min per filtration
step. The treatment with 0.1 g H-300 plus 0.1 g H-400 gelled and took longer to filter completely.
In addition, debris-containing samples are expected to have longer filtration times, but were not
tested in this effort.
After the wash step, the vial bottom was capped and 5 mL YPEB were added. Due to the
experiment timing, a To aliquot could not be taken; however, T2 samples were taken after 2-hr
incubation for both culturing and RV-PCR analysis. The Autovial samples were incubated at 30°C
with shaking at 180 rpm, and after 24 hr another 1 mL aliquot was removed (T24 aliquot). Aliquots
from both time-points were extracted for DNA using the Promega MagneSil reagents, and
undiluted and 10-fold diluted extracts were analyzed using the YC2 assay in triplicate with
Universal reagents and Standard cycling conditions (45 amplification cycles).
Since results showed that Ct values for 10-fold diluted DNA extracts were lower than those from
undiluted extracts (suggesting PCR inhibition), only results for 10-fold diluted extracts are shown
in Table 13. The 0.1 g MG treatment compared most favorably to the control treatment in terms
of average T24 Ct values (15.8 and 14.9, respectively). The MG treatment also showed good
filtration behavior, allowing filtration similar to the case without MG material (i.e., control).
Limited plate count analysis (single replicates) at T24 showed approximately 2 x 108 CFU/sample
for the 0.1 g MG sample. This was less than the control although accurate counts could not be
obtained from either treatment since the correct dilution was not plated and colonies were too
numerous to count accurately. No counts from the DE samples were obtained due to the presence
of contaminating colonies (background organisms).
A follow-up plating experiment with MG material showed that it was sterile. Therefore, the MG
material represented the best candidate material for the MF approach to concentrate Y pestis and
possibly other vegetative cells prior to RV-PCR analysis. However, MF was not pursued further
mostly due to variation in SAP behavior from one experiment to the next with regard to physical
structure and filtration properties. It was difficult to reproducibly prepare the materials to allow
rapid filtration with the current setup; therefore use of this approach for cell concentration could
add significantly to the sample processing time. Slight variation in weights (or particle size
distributions of the materials) applied to the Autovials led to gel formation and poor filtration
whereas other times SAPs remained more dispersed and showed rapid filtration. The variability in
filtration behavior would make it difficult to employ this approach operationally, such that more
investigation would be required. It is also possible that the material amount and method could not
be sufficiently standardized for high throughput sample processing.
35
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Table 13 Real-time PCR Results for RV-PCR Analysis* of Y. pestis C092 pgm Cells
(~4 x io4) Collected by a Modified Filtration Approach
Modified
Filtration
PCR
Replicate
YC2 Assay Ave. Ct (SD)*
act (t2-t24)
Treatment**
T 2
t24
1
31.4
19.4
0.1 g DE
2
31.2
18.9
12.1
3
31.1
19.2
Ave (SD)
31.3 (0.1)
19.2 (0.2)
1
32.4
23.2
0.2 g DE
2
32.8
23.2
9.5
3
33.2
23.4
Ave (SD)
32.8 (0.4)
23.3 (0.1)
1
30.7
18.3
0.1 g DE + 0.1 g
2
30.8
18.4
12.5
MG
3
30.9
18.3
Ave (SD)
30.8 (0.1)
18.3 (0.1)
1
29.5
15.7
0.1 g MG
2
29.5
15.8
13.7
3
29.6
15.7
Ave (SD)
29.5 (0.1)
15.8 (0.1)
1
31.8
18.0
0.1 g H-300 +
2
32.2
18.2
14.1
0.1 g H-400
3
32.3
17.8
Ave (SD)
32.1 (0.2)
18.0 (0.2)
1
30.2
14.9
Control
2
30.5
14.9
15.4
3
30.2
14.9
Ave (SD)
30.3 (0.2)
14.9 (0.1)
* 10-fold diluted DNA extracts were analyzed. Universal Master Mix and Standard cycling conditions were used.
** Average (Ave) and standard deviation (SD) from triplicate PCR analyses. DE = diatomaceous earth; MG =
(R)
Miracle Gro (100% acrylainide); H-300 and H-400 = superabsorbent polymers (JRM Chemical, Inc.).
36
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4.2.8. Evaluation of Immunomagnetic Separation for Concentration of Y. pestis Cells from
Larger Volume Water Samples
This proof-of-principle study was conducted using IMS to capture of Y pestis cells from PBS
solutions as a surrogate for water samples. The Pathatrix system was used to concentrate Y pestis
cells from -30 mL prior to RV-PCR analysis. Anti-K pestis polyclonal antibody from the Critical
Reagents Program (CRP) through BEI Resources, Inc. (BEI Cat. No. DD-514; CRP Cat. No. AB-
G-YERS) was used to coat the beads. Pathatrix beads were coated following the manufacturer's
directions using recommended antibody concentrations. Two experiments were conducted using
RV-PCR analysis of IMS beads containing Y pestis cells captured from suspensions and limited
plating was conducted to estimate cell recovery efficiency using IMS.
Y pestis C092 pgm cells were grown overnight in YPEB, washed in PBS and prepared to ~ 107
CFU/mL. Ten-fold serial dilutions were made in PBS and cells were added at three levels (-50,
-500, and -5000 CFU determined from reference plating) to 30 mL PBS samples (as a surrogate
for actual water samples). Counts were actually 54-5400 and 56-5600 for the first and second
experiments, respectively. The cell levels were tested in triplicate and a negative control IMS
sample without cells was also processed using 30 mL PBS. The recommended amount of antibody-
coated Pathatrix beads was used (50 |iL) for each sample or control. The recovered beads from
IMS were resuspended in 100 [xL PBS with 20 [xL used to prepare dilutions for plating analysis,
and the remaining 80 [xL used in an RV-PCR experiment. Specifically, this aliquot (80 [xL) was
added to 3 mL YPEB in a 48-well plate for RV-PCR analysis. Controls had 3 mL of the original
cell suspension added to wells, corresponding to the same number of cells processed by IMS. In
addition, two negative controls were included in the 48-well plate by adding 3 mL PBS. At To, the
well contents were pipet-mixed and a 0.5 mL aliquot was removed and processed for DNA and
subsequent real-time PCR analysis. After 24 hr incubation, another 0.5 mL aliquot was obtained
and processed for PCR analysis. DNA was extracted using the modified Promega Magnesil
protocol and extracts were analyzed using the YC2 (chromosomal) assay without dilution.
The RV-PCR results from the first and second replicate experiments are shown in Table 14 and
Table 15, respectively. For both experiments, 3 of 3 were positive for viable Y pestis for -500 and
-5000 cells processed by IMS, however, either 2 of 3 or 1 of 3 replicates were positive for -50
cells processed by IMS for the first and second replicate experiments, respectively. This is
contrasted with the control treatments (which were not diluted to 33 mL and processed by IMS
prior to RV-PCR), which had 3 of 3 positive by RV-PCR for all three cell levels. Similar trends
were observed between the two experiments. In general there was about a two Ct difference in
ACt values between IMS-treated cells and control cells at the -500 and -5000 cell levels (the
difference was < 1 cycle for the 500-cell level for the first experiment), although the T24 Ct values
differed by -4-7 cycles for treated and control cells. There were greater differences between the
IMS and control treatments for the -50 cell level.
Limited culture analysis of Pathatrix beads on TBA plates was performed in parallel in order to
obtain some data on cell recovery using IMS. For the first experiment, culture results from the
-5000 cell level showed variable recoveries ranging from -2-37%. For the second experiment,
more data were obtained showing that recoveries ranged from -3-14% for both the -500 and
-5000 cell levels. However, when the remaining cell suspension from the Pathatrix instrument was
plated (representing cells not captured on beads) and used to determine the cells captured (by
37
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difference), the recoveries were higher based on this analysis with an average of 36.3 ± 10.9%
recovered for the different cell levels. Based on this data, it is possible that plating of beads
underestimated the actual cell recovery; however, there were clearly significant losses of cells
based on plating the remaining suspension. Furthermore, when starting with -50 cells all three
replicates were not detected, whereas starting cell levels down to 10 CFU per sample have
previously been detected by RV-PCR at 24 hr. It is also possible that cells captured onto beads
propagated more slowly (with a possible lag period) compared to cells not treated with this method.
Table 14 Real-time PCR Results from RV-PCR Analysis of Water Samples Containing
Different Levels of Y. pestis C092 pgm" Cells Processed by IMS - YC2 Assay
Sample Type
Sample
Replicate
Ave. CT (SD)*
ACt
Positive
Replicates
To
t24
-5000 cells IMS
1
NDT
24.6(0.1)
20.4
3 of 3
2
39.1 (0.4)
22.4 (0.1)
16.7
3
NDT
23.2 (0.1)
21.8
Ave (SD)
43.0 (3.0)
23.4 (1.0)
19.6 (2.6)
~5000 cells control
1
34.5 (0.3)
18.6(0.2)
16.0
3 of 3
2
34.9(0.4)
18.3 0.2)
16.6
3
35.8 (0.4)
19.7(0.1)
16.1
Ave (SD)
35.1 (0.6)
18.9 (0.7)
16.2 (0.4)
-500 cells IMS
1
NDT
28.2 (0.2)
16.8
3 of 3
2
NDT
26.4 (0.1)
18.6
3
41.9(0.2)**
27.5 (0.1)
14.3
Ave (SD)
44.0 (1.6)
27.4 (0.8)
16.6 (2.2)
~500 cells control
1
37.8 (0.9)
20.5 (0.1)
17.3
3 of 3
2
37.5 (0.8)
20.5 (0.1)
17.0
3
38.7(0.9)
20.7(0.1)
18.0
Ave (SD)
38.0 (0.9)
20.6 (0.1)
17.4 (0.6)
-50 cells IMS
1
NDT
NDT
0.0
2 of 3
2
NDT
31.3 (0.1)
13.7
3
41.7(0.2)**
28.9(0.1)
12.8
Ave (SD)
43.4 (2.3)***
30.1 (1.6)***
13.3 (0.7)***
-50 cells control
1
NDT
22.8 (0.2)
22.2
3 of 3
2
NDT
24.4 (0.1)
20.6
3
NDT
24.6(0.1)
20.4
Ave (SD)
NDT
23.9 (0.9)
21.1 (1.0)
Negative Control for IMS
NDT
NDT
0.0
Oof 1
Negative control for
48-well plate
1
NDT
NDT
0.0
Oof 2
2
41.0(0.7)**
NDT
-4.0
Ave (SD)
43.0 (2.2)
NDT
-2.0 (2.8)
* Average (Ave) and standard deviation (SD) are from triplicate PCR analyses per sample replicate. Universal
Master Mix and Standard cycling conditions were used.
** Values are from two PCR replicates; the third replicate was non-detect.
*** Values are from two sample replicates; the third replicate was non-detect (negative).
NDT = Non-Detect. NDT set to 45 to calculate ACt.
38
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Table 15 Real-time PCR Results from RV-PCR Analysis of Water Samples Containing
Different Levels of Y. pestis C092 pgm" Cells Processed by IMS - YC2 Assay (Replicate
Experiment)
Sample Type
Sample
Replicate
Ave. CT (SD)*
ACx
Positive
Replicates
To
t24
-5000 cells IMS
1
NDT
23.0 (0.2)
22.0
3 of 3
2
NDT
21.7 (0.2)
23.3
3
NDT
21.7 (0.1)
23.3
Ave (SD)
NDT
22.1 (0.7)
22.9 (0.8)
~5000 cells control
1
34.9(0.7)
17.7 (0.1)
17.2
3 of 3
2
39.2 (0.9)
18.7 (0.1)
20.5
3
40.1 (0.9)
18.7 (0.1)
21.4
Ave (SD)
38.1 (2.5)
18.4 (0.5)
19.7 (2.2)
-500 cells IMS
1
NDT
28.3 (0.2)
16.7
3 of 3
2
NDT
28.3 (0.2)
16.7
3
NDT
23.2 (0.1)
21.8
Ave (SD)
NDT
26.6 (2.6)
18.4 (3.0)
~500 cells control
1
38.7(0.6)
21.2 (0.1)
17.5
3 of 3
2
37.4 (0.3)
21.3 (0.1)
16.1
3
38.8 (2.1)
21.4 (0.1)
17.4
Ave (SD)
38.3 (1.3)
21.3 (0.1)
17.0 (1.0)
-50 cells IMS
1
NDT
NDT
0.0
1 of 3
2
NDT
NDT
0.0
3
NDT
32.0 (0.1)
13.0
Ave (SD)
NDT
NA
NA
-50 cells control
1
NDT
23.9 (0.1)
21.1
3 of 3
2
NDT
23.9 (0.1)
21.1
3
NDT
24.0 (0.1)
21.0
Ave (SD)
NDT
23.9 (0.1)
21.1 (0.1)
Negative control for IMS
NDT
NDT
0.0
Oof 1
Negative control for
48-well plate
1
NDT
NDT
0.0
Oof 2
2
NDT
NDT
0.0
Ave (SD)
NDT
NA
0.0
* Average (Ave) and standard deviation (SD) are from triplicate PCR analyses per sample replicate. Universal
Master Mix and Standard cycling conditions were used.
NA = Not Applicable; NDT = Non-Detect. NDT set to 45 to calculate ACt.
This preliminary analysis of IMS integrated with RV-PCR showed relatively poor recovery of
cells such that additional experiments with IMS were not conducted. It is possible that use of
affinity-purified antibody could have enabled better cell recoveries (although these were not
available for this effort). The cost, availability, and reproducibility of the IMS approach also need
to be considered, especially for operational use for detection of viable Y pestis from water samples.
In this case, IMS would also increase the overall time to results from about 36 hr to 40 hr for 48
samples, since each batch of five samples (up to 60 mL) takes about 25 min (15 min on the
Pathatrix instrument and 10 min sample preparation and recovery).
39
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4.3 TASK 3: Further development and optimization of RV-PCR protocols for Y. pestis
4.3.1. Objectives
With the optimized protocol resulting from improvements in cell growth procedures (Task 2) and
DNA purification/concentration procedures (Task 1), a shorter incubation period of 24-hr was
proposed. The main objective of this task was then to evaluate whether the shorter incubation
resulted in positive detection of viable Y pestis cells even for the types of complex samples
expected. The RV-PCR protocol for Y pestis had not previously been tested with challenges
including potential PCR and growth inhibitors. Furthermore, method evaluation in a background
of dead, target cells was required to determine how the method would work in real-world
decontamination or natural degradation scenarios. With these challenges, it was important to
evaluate the incubation period and LOD in order to assess method performance with regard to the
types of water samples that could be analyzed using this method. Therefore, the optimized
protocols from Tasks 1 and 2 were used to confirm the RV-PCR method incubation and establish
the method LOD for different challenges including potential chemical and biological interferences.
4.3.2. Evaluation of RV-PCR Method Performance with Complex Water Samples
In this task, water samples with iron sulfate and humic acids as chemical interferences were
evaluated. These were selected since iron and humics are often mentioned as PCR inhibitors and
the goal was to ensure that their presence did not negatively impact either Y pestis growth or PCR
analysis of DNA extracted from cells. Iron sulfate and humic acid levels were selected that were
representative (although at the high end) of levels expected from actual water samples (NRC, 1979;
WHO, 1996). In addition, water samples containing native AZ Test Dust representing both
chemical (metals, oxides) and biological (live, non-target cells) interferences were also evaluated.
The characterization data is included in Materials and Methods Section 2.4.
The Y pestis RV-PCR method was evaluated with water samples (using phosphate-buffered
saline) containing complex backgrounds of live non-target spores/cells (non-autoclaved AZ Test
Dust, 4 mg/mL), or humic acids (50 (j.g/mL) plus iron (10 [j,g/mL Fe as FeSO/t). These samples
were compared with controls lacking the challenge material to determine the effect of biological
and chemical challenges on RV-PCR method performance for Y pestis. An overnight culture of
Y pestis C092 was harvested by centrifugation, washed with PBS, and diluted to 0.1 OD600 with
PBS (approximately 1 x 107 CFU/mL). Y pestis C092 cells were serially diluted and added at -18
or 180 CFU/mL (from reference plating). Cells in PBS were reconstituted by adding 10X YPEB
and 3 mL were added to each well. A 0.5 mL To aliquot was removed, centrifuged at 20,800 RCF
for 10 min at 4°C, 300 [iL were removed, and the resulting pellet was stored at -20°C until pellets
were extracted for DNA. The remaining culture was incubated at 30°C with shaking at 180 rpm
for 24 hr, after which a 0.5 mL T24 aliquot was removed and processed as for the To aliquot.
Aliquots were extracted for DNA using Promega Magnesil reagents and PCR analysis was
conducted using the YC2 assay.
For the 100-cell level (Table 16), the data showed that there was no PCR inhibition for the control
treatment with similar ACt values for undiluted and 10-fold diluted extracts; however, the
40
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treatment with iron and humic acid showed inhibition for 1 of the 3 replicates, while the 10-fold
diluted extract had similar data to the control treatment (i.e., elimination of PCR inhibition). For
the treatment with native (un-autoclaved) test dust, about 30% lower ACt values were observed
(Average [Ave.] ACt = 8.8 ± 3.6) than those for the other treatments (Ave. ACt = 13.1 ±0.3 and
12.5 ± 0.5 for the control and Fe/Humic treatments, respectively). This was likely due to growth
inhibition from the indigenous organisms in the test dust, which includes faster growing genera
such as Bacillus. Plating could not be conducted at the end of incubation since Y pestis colonies
would not easily be detected in the background colony growth. The ACt values were > 6 (the
criteria set-up for detection of live cells in samples) for only 2 of the 3 replicates for native ATD
suggesting a longer incubation period may be needed for detection of low cell levels for these types
of samples.
The same trends were observed for the lower cell level (-18 CFU/mL; Table 17) with lower ACt
values for the native test dust treatment; however in this case, the ACt values were significantly
lower than 6, ranging from 1.3 to 4.4 for the 10-fold diluted extracts showing that positive detection
could not be achieved for these low starting cell levels in the presence of background organisms
at least after 24 hr. As for the 100-cell level samples, the 10-fold dilution gave consistently higher
ACt values (that met the requirement for positive detection) than the undiluted extracts for the
Fe/Humic treatment, showing PCR inhibition in undiluted samples.
A replicate evaluation was conducted for the Y pestis RV-PCR method with water samples (using
PBS) containing challenge materials at the same concentrations, AZ Test Dust (4 mg/mL), or
humic acids (50 (j,g/mL) plus iron (10 |ig/mL Fe as FeSC>4). These treatments (in triplicate) were
also compared with triplicate controls lacking the challenge material. Y pestis cells were prepared
as described for the previous experiment, and added at -10 or 100 CFU/mL per sample well
(determined from reference plating). Aliquots were removed and subjected to DNA extraction and
PCR analysis as described previously.
For the 100-cell level (Table 18), as for the initial experiment the data showed that there was little
to no PCR inhibition for the control treatment with similar ACt values for undiluted and 10-fold
diluted extracts; however, the treatment with iron and humic acid showed inhibition for 1 of the 3
replicates for undiluted extracts, while the 10-fold diluted extract had similar data to the control
treatment. However, contrary to the first replicate experiment (Table 17), the treatment with native
(un-autoclaved) test dust showed similar ACt values to the control treatment for either undiluted
or 10-fold diluted extracts. Unlike the first replicate experiment, higher To Ct values were
observed, which led to greater ACt values since T24 Ct values were comparable between
experiments. Although, the replicate experiments were conducted the same with regard to Y pestis
cell preparation, it appeared that the first experiment had a higher concentration of dead cells that
led to lower To Ct values. The first experiment also used slightly higher starting cell concentrations
although the difference was only about 2-fold higher.
Similar trends were observed for the lower cell level (-10 CFU/mL; Table 19); however in this
case there were lower ACt values for the native test dust treatment compared to the control
treatment especially for the 10-fold diluted extracts. It should also be noted that 1 of 3 control
replicates showed high T24 Ct values (-32-33) likely due to poor growth and/or operator error in
DNA extraction or PCR analysis for this replicate. Unlike the previous experiment, the ACt values
41
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for the native test dust treatment still met the criterion for positive detection for all but one replicate
for 10-fold dilution only (for the undiluted extract, the ACt value was 12.2). For the Fe/humics
treatment, only 1 of 3 replicates showed PCR inhibition although this was resolved with 10-fold
dilution. Together these results suggest that debris such as the reference test dust could cause
growth inhibition due to the presence of indigenous organisms such as faster growing Bacillus spp.
The test dust was shown to contain Bacillus and other bacteria as well as fungal spores (Rose et
al., 2011). However, at these low cell levels, the RV-PCR method still showed the ability to
accurately detect live cells in complex backgrounds, with consistent detection at the 100-cell level.
The negative controls were non-detect for all replicates at both time points (data not shown).
The first experiment had lower To Ct values, which impacted the ACt values; the T24 Ct values
were comparable between experiments. Although the experiments were conducted the same way
with regard to cell propagation and preparation of the inoculum, the first experiment appeared to
have a higher concentration of dead cells, which likely contributed to lower To Ct values, although
this was not measured directly. The cells used as inoculum were obtained from an overnight culture
that was washed to remove spent broth but dead cells could not be removed. Regardless, the
experiments showed that Fe/humic acid did not impact the method incubation period and LOD;
however, for low cell numbers (-10-20 cells per sample) especially with high dead cell
concentrations, detection after 24-hr incubation could be inconsistent such that a longer incubation
period (i.e., 36 hr) could be warranted.
42
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Table 16 Real-time PCR Results for RV-PCR DNA Extracts from -180 Y. pestis C092
Cells Added to 48-Well Plates With and Without Chemical or Biological Backgrounds
Sample Replicate
- PCR Replicate
YC2 Assay Ave. Ct (SD)*
ACt (T
-t
r*
H
1
O
Treatment
Undiluted
10-Fold Dilution
Undiluted
10-Fold
To
T24
To
T24
Dilution
1 - 1
34.6
22.4
37.9
25.3
1-2
34.0
22.1
38.2
25.0
12.2
12.9
1-3
34.6
22.0
38.1
25.2
Ave (SD)
34.4 (0.4)
22.2 (0.2)
38.1 (0.2)
25.2 (0.2)
2-1
34.0
22.1
37.5
24.8
2-2
33.8
21.7
38.2
24.8
12.1
13.1
Control
2-3
33.9
21.6
38.1
24.9
Ave (SD)
33.9 (0.1)
21.8 (0.2)
38.0 (0.4)
24.9 (0.1)
3 - 1
33.8
21.6
37.3
24.3
3-2
33.3
21.2
37.7
24.2
12.2
13.4
3-3
33.5
21.2
38.2
24.3
Ave (SD)
33.5 (0.2)
21.3 (0.2)
37.7 (0.4)
24.3 (0.1)
Overall Ave (SD)
33.9 (0.5)
21.8 (0.4)
37.9 (0.4)
24.8 (0.4)
12.1 (0.1)
13.1 (0.3)
1 - 1
39.2
27.4
38.2
25.3
1-2
38.5
26.0
38.2
25.3
12.5
12.8
1-3
38.8
25.5
38.0
25.3
Ave (SD)
38.8 (0.3)
26.3 (1.0)
38.1 (0.1)
25.3 (0.1)
2-1
36.6
40.9
40.2
27.4
2-2
35.9
39.0
38.9
27.5
-3.0
11.9
Fe/Humics
2-3
35.8
37.4
39.2
27.5
Ave (SD)
36.1 (0.5)
39.1 (1.8)
39.4 (0.7)
27.5 (0.1)
3 - 1
36.1
26.1
37.7
25.1
3-2
36.3
25.3
38.0
25.1
10.6
12.8
3-3
35.9
25.1
38.0
25.1
Ave (SD)
36.1 (0.2)
25.5 (0.5)
37.9 (0.2)
25.1 (0.1)
Overall Ave (SD)
37.0 (1.4)
30.3 (6.7)
38.5 (0.8)
26.0 (1.2)
6.7 (8.5)
12.5 (0.5)
1 - 1
34.6
31.2
37.7
32.2
1-2
35.0
31.0
37.9
32.2
3.9
5.7
1-3
34.8
30.6
38.4
32.4
Ave (SD)
34.8 (0.2)
30.9 (0.3)
38.0 (0.4)
32.3 (0.1)
2-1
37.1
33.6
39.9
32.4
Native
2-2
37.2
32.2
NDT
32.2
4.7
12.7
ATD
2-3
37.3
31.7
NDT
32.3
Ave (SD)
37.2 (0.1)
32.5 (1.0)
NDT
32.3 (0.1)
3 - 1
36.2
33.2
39.9
32.0
3-2
36.6
32.3
38.4
32.1
3.8
7.9
3-3
36.2
31.9
41.9
32.4
Ave (SD)
36.3 (0.2)
32.5 (0.7)
40.1 (1.8)
32.2 (0.2)
Overall Ave (SD)
36.1 (1.1)
32.0 (1.0)
41.0 (3.3)
32.3 (0.1)
4.1 (0.5)
8.8 (3.6)
* Average (Ave) and standard deviation (SD) are from Universal reagents and Standard cycling conditions.
ATD = Arizona Test Dust. NDT = Non-detect.
43
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Table 17 Real-time PCR Results for RV-PCR DNA Extracts from ~18 Y. pestis C092 Cells
Added to 48-Well Plates With and Without Chemical or Biological Backgrounds
Sample Replicate
- PCR Replicate
YC2 Assay Ave. Ct (SD)*
ACt (T
-t
r*
H
1
O
Treatment
Undiluted
10-Fold Dilution
Undiluted
10-Fold
To
T24
To
T24
Dilution
1-1
38.4
25.4
38.1
28.1
1-2
38.1
25.3
38.1
27.9
12.8
10.4
1-3
37.9
25.1
39.3
28.2
Ave (SD)
38.1 (0.3)
25.3 (0.2)
38.5 (0.7)
28.1 (0.2)
2-1
34.1
25.3
37.7
27.7
2-2
34.0
24.8
37.8
27.7
9.1
10.1
Control
2-3
34.0
24.7
38.2
27.9
Ave (SD)
34.0 (0.1)
24.9 (0.3)
37.9 (0.3)
27.8 (0.1)
3-1
33.6
27.0
38.1
29.7
3-2
33.5
26.6
37.3
29.6
6.9
9.2
3-3
33.5
26.4
41.3
29.7
Ave (SD)
33.6 (0.1)
26.7 (0.3)
38.9 (2.1)
29.7 (0.1)
Overall Ave (SD)
35.2 (2.2)
25.6 (0.9)
38.4 (1.2)
28.6 (0.9)
9.6 (3.0)
9.8 (0.6)
1-1
35.1
28.2
37.4
27.6
1-2
34.9
28.4
38.8
27.6
7.0
10.3
1-3
35.3
27.7
37.6
27.6
Ave (SD)
35.1 (0.2)
28.1 (0.4)
37.9 (0.7)
27.6 (0.1)
2-1
41.0
40.3
38.4
27.1
2-2
40.0
37.8
39.7
27.1
2.4
11.5
Fe/Humics
2-3
40.0
35.7
38.0
27.2
Ave (SD)
40.3 (0.6)
37.9 (2.3)
38.7 (0.9)
27.2 (0.1)
3-1
35.5
28.6
39.0
28.8
3-2
34.9
28.1
40.4
28.7
7.0
10.4
3-3
35.3
27.8
38.1
28.8
Ave (SD)
35.2 (0.3)
28.2 (0.4)
39.2 (1.1)
28.8 (0.1)
Overall Ave (SD)
36.9 (2.6)
21.4 (5.0)
38.6 (1.0)
27.9 (0.7)
5.5 (2.7)
10.7 (0.7)
1-1
35.9
33.8
38.5
35.0
1-2
35.3
33.2
38.3
35.2
2.4
3.3
1-3
36.2
33.2
38.2
35.0
Ave (SD)
35.8 (0.5)
33.4 (0.3)
38.3 (0.1)
35.0 (0.1)
2-1
35.8
35.8
37.2
37.4
Native
2-2
35.8
35.5
39.1
37.1
0.1
1.3
ATD
2-3
35.4
35.5
38.8
36.8
Ave (SD)
35.7 (0.2)
35.6 (0.2)
38.4 (1.0)
37.1 (0.3)
3-1
36.0
32.9
39.8
34.6
3-2
35.6
32.5
38.8
34.3
3.3
4.4
3-3
36.1
32.3
38.0
34.5
Ave (SD)
35.9 (0.3)
32.6 (0.3)
38.9 (0.9)
34.5 (0.2)
Overall Ave (SD)
35.8 (0.3)
33.9 (1.4)
38.5 (0.7)
35.5 (1.2)
1.9 (1.7)
3.0 (1.6)
* Average (Ave) and standard deviation (SD) are from Universal reagents and Standard cycling conditions.
ATD = Arizona Test Dust.
44
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Table 18 Real-time PCR Results for RV-PCR DNA Extracts from -100 Y. pestis C092
Cells Added to 48-Well Plates With and Without Chemical or Biological Backgrounds
Sample Replicate
- PCR Replicate
YC2 Assay Ave. Ct (SD)*
ACt (T0-T24)
Treatment
Undiluted
10-Fold Dilution
Undiluted
10-Fold
To
T24
To
T24
Dilution
1-1
38.6
24.2
NDT
28.3
1-2
39.6
23.3
NDT
28.1
15.2
16.8
1-3
38.3
23.3
40.2
28.4
Ave (SD)
38.8 (0.7)
23.6 (0.5)
NDT
28.2 (0.2)
2-1
37.0
23.7
39.7
21.2
2-2
36.7
25.2
NDT
24.2
12.7
16.2
Control
2-3
37.1
24.0
39.1
24.3
Ave (SD)
37.0 (0.2)
24.3 (0.8)
39.4 (0.4)**
23.2 (1.8)
3-1
38.0
22.6
38.1
25.8
3-2
37.1
23.1
NDT
26.8
14.9
12.9
3-3
38.6
23.4
41.0
27.2
Ave (SD)
37.9 (0.8)
23.0 (0.4)
39.5 (2.1)**
26.6 (0.7)
Overall Ave (SD)
37.9 (0.9)
23.6 (0.8)
41.3 (3.0)
26.0 (2.4)
14.3 (1.4)
15.3 (2.1)
1-1
NDT
28.4
NDT
30.0
1-2
41.0
27.5
39.8
31.0
17.3
14.6
1-3
NDT
27.2
NDT
30.4
Ave (SD)
NDT
27.7 (0.6)
NDT
30.4 (0.5)
2-1
44.7
33.4
NDT
31.4
Fe/
Humics
2-2
43.3
NDT
41.7
26.2
12.7
13.1
2-3
42.8
28.8
41.1
27.4
Ave (SD)
43.8 (1.3)**
31.1 (3.3)**
41.4 (0.4)**
28.3 (2.8)
3-1
42.1
31.1
40.1
28.3
3-2
41.6
42.8
40.3
26.6
3.3
13.4
3-3
41.7
41.8
NDT
25.7
Ave (SD)
41.8 (0.3)
38.5 (6.5)
40.2 (0.1)**
26.8 (1.3)
Overall Ave (SD)
43.5 (1.5)
32.4 (6.0)
42.2 (2.2)
28.5 (2.2)
11.1 (7.1)
13.7 (0.8)
1-1
NDT
31.6
40.6
32.5
1-2
41.8
31.0
NDT
32.4
13.7
12.2
1-3
NDT
31.4
NDT
33.5
Ave (SD)
NDT
31.3 (0.3)
NDT
32.8 (0.4)
2-1
43.1
27.4
NDT
29.6
Native
2-2
NDT
26.7
40.5
29.5
15.9
15.3
ATD
2-3
42.7
26.8
NDT
30.1
Ave (SD)
42.9 (0.3)**
27.0 (0.4)
NDT
29.7 (0.3)
3-1
NDT
30.0
NDT
32.5
3-2
NDT
29.0
NDT
32.1
15.5
12.6
3-3
NDT
29.6
NDT
32.6
Ave (SD)
NDT
29.5 (0.5)
NDT
32.4 (0.2)
Overall Ave (SD)
44.3 (1.1)
29.2 (1.9)
NDT
31.6 (1.5)
15.1 (1.2)
13.4 (1.7)
* Average (Ave) and standard deviation (SD) are from Universal reagents and Standard cycling conditions.
** Values are from two PCR replicates; the third replicate was non-detect.
NDT = Non-detect. NDT set to 45 to calculate ACt.ATD = Arizona Test Dust.
45
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Table 19 Real-time PCR Results for RV-PCR DNA Extracts from ~10 Y. pestis C092 Cells
Added to 48-Well Plates With and Without Chemical or Biological Backgrounds
Sample Replicate
- PCR Replicate
YC2 Assay Ave. Ct (SD)*
ACt (T0-T24)
Treatment
Undiluted
10-Fold Dilution
Undiluted
10-Fold
To
T24
To
T24
Dilution
1-1
39.1
24.6
NDT
29.0
1-2
39.4
24.3
NDT
30.0
14.7
15.2
1-3
38.9
24.3
NDT
30.3
Ave (SD)
39.1 (0.3)
24.4 (0.2)
NDT
29.8 (0.7)
2-1
40.9
33.2
NDT
34.1
2-2
40.5
32.4
NDT
33.7
7.9
11.1
Control
2-3
40.3
32.5
NDT
33.9
Ave (SD)
40.6 (0.3)
32.7 (0.5)
NDT
33.9 (3.3)
3-1
NDT
23.9
NDT
27.9
3-2
NDT
23.5
NDT
27.9
21.4
17.0
3-3
NDT
23.3
NDT
28.3
Ave (SD)
NDT
23.6 (0.3)
NDT
28.0 (0.2)
Overall Ave (SD)
41.6 (2.7)
26.9 (4.4)
NDT
30.6 (3.1)
14.7 (6.8)
14.4
1-1
42.4
34.8
NDT
31.2
1-2
42.2
32.4
NDT
31.0
9.1
13.9
1-3
NDT
32.3
NDT
31.1
Ave (SD)
42.3 (0.1)**
33.2 (1.4)
NDT
31.1 (0.1)
2-1
44.1
37.7
NDT
30.3
2-2
NDT
31.8
NDT
30.3
11.1
14.7
Fe/Humics
2-3
NDT
32.2
NDT
30.2
Ave (SD)
NDT
33.9 (0.2)
NDT
30.3 (0.1)
3-1
NDT
NDT
43.5
28.2
3-2
NDT
NDT
41.1
28.2
0.0
14.0
3-3
NDT
39.7
NDT
28.3
Ave (SD)
NDT
NDT
42.3 (1.7)**
28.3 (0.1)
Overall Ave (SD)
44.1 (1.4)
37.3 (5.8)
44.1 (1.6)
29.9 (1.3)
6.7 (5.9)
14.2
1-1
NDT
34.2
40.0
36.2
1-2
NDT
33.7
NDT
36.1
12.2
4.1
1-3
43.5
33.5
40.1
35.3
Ave (SD)
NDT
33.8 (0.4)
40.0 (0.1)**
35.9 (0.5)
2-1
NDT
34.5
NDT
36.9
Native
2-2
NDT
34.1
NDT
38.5
10.8
7.4
ATD
2-3
NDT
34.0
NDT
37.5
Ave (SD)
NDT
34.2 (0.2)
NDT
37.6 (0.8)
3-1
41.3
32.9
NDT
35.8
3-2
39.1
32.9
41.6
35.4
7.8
9.5
3-3
41.3
32.3
NDT
35.4
Ave (SD)
40.5 (1.3)
32.7 (0.4)
NDT
35.5 (0.3)
Overall Ave (SD)
43.5 (2.3)
33.6 (0.7)
43.3 (2.5)
36.3 (1.1)
10.3 (2.2)
7.0 (2.7)
* Average (Ave) and standard deviation (SD) are from Universal reagents and Standard cycling conditions.
** Values are from two PCR replicates; the third replicate was non-detect.
NDT = Non-detect. NDT set to 45 to calculate ACt. ATD = Arizona Test Dust.
46
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4.3.3. Evaluation of RV-PCR Method Performance in a Dead Y. pestis Cell Background
The Y. pestis RV-PCR method with 24-hr incubation period was evaluated for application to post-
decontamination scenarios where low levels of live target cells must be detected in samples with
high concentrations of dead target cells. Initially, methods for generating a background of dead
cells were investigated such that the cells remained intact. Intact, dead cells represented the most
challenging case for testing the RV-PCR method. Different disinfection methods included
autoclaving, antibiotic exposure, and isopropanol exposure. After generating dead cell
populations, sterility was confirmed by plating analysis using 30% volume of the cell suspension
(300 |iL from total volume of 1 mL with ~5 x 107 CFU). The dead cell concentrations ranged from
102 to 106 cells per sample.
As described in Materials and Methods Section 2.6, the pgm strain was used for IPA exposure,
using protocols from generation of dead cells for microscopy and flow cytometry as referenced
(i.e., Live/Dead® BacLight™ Bacterial Viability Kit, Molecular Probes, Inc.). Initially, a 1-hr
exposure to 70% IPA was tested with ~ 1 x 109 CFU/mL; however, only about 6-1 og kill was
achieved based on serial dilution and plating. After discussion with the EPA Technical Lead, the
culture conditions were changed to be more controlled (synchronized) and exposures were
conducted with fewer cells, ~5 x 107 CFU/mL (early log phase) for both 1 and 2 hr with mixing
every 30 min. These conditions produced completely dead cell suspensions for both exposure
times based on plating triplicate 0.1 mL aliquots (detection limit of ~3 - 4 CFU/mL).
Prior to RV-PCR experiments, IPA-killed cells were tested for DNA content by heat lysis and PCR
analysis. Since previous PCR analysis showed that the pgnT strain was also reactive with the YC2
(as well as YpPl, and YpMTl) assays, this allowed use of the YC2 assay to evaluate whether the
disinfection method led to loss of DNA from cells. A flow chart for generation of dead cells and
PCR analysis of different components during the preparation of the IPA-killed cell suspension is
shown in Figure 8. Control cell suspensions treated with PBS instead of IPA were processed and
analyzed for DNA content in parallel.
The Ct values were comparable between IPA-killed cells and control cells and even showed lower
average Ct values for IPA-killed cells (Table 20). This trend was consistent for heat lysates from
cell suspensions and cell pellets as well as from supernatants. The PCR data from supernatants
showed loss of DNA for both treated and untreated cells although values suggested about 2-log
less DNA in supernatants compared to the other fractions. Similar analysis was conducted 9 days
later. The data showed no significant differences between PCR results for the different components
for IPA-killed and control cell suspensions with p-values ranging from 0.1 - 0.6 (Student's two-
tailed, paired T-test). In addition, cells tested in the same way up to 40 days after initial preparation
showed similar results for heat lysates of cell suspensions and supernatants. These data suggested
that dead cells remained intact and stable over time while stored at 4°C, thus providing the most
challenging test case for a dead cell background for RV-PCR (highest levels of DNA from dead
cells). The 2-hr IPA-exposed cells were used in RV-PCR experiments and tested by PCR for loss
of DNA prior to each experiment (i.e., cell used up to 40-days after exposure and preparation).
47
-------
Generation of IPA-
Kvaluation of IPA-killed
killed Y. pestis cells cell integrity/DNA loss
Heat Lysis
PCR Analysis
PCR Analysis
PCR Analysis
Cell pellet
Cell pellet
Cell pellet
PCR Analysis
PCR Analysis
Heat Lysis
Supernatant #2
Supernatant #1
Centrifuge cells
Centrifuge cells
Wash cells
with PBS
Heat Lysate
from Cell Pellet
IPA-Killed Cell
Suspension
20 mL Y. pestis cells
(107 CFU/mL)
Centrifuge cells as for
RV-PCR aliquots
Expose cells to 70%
IPA for 2 hr
Heat Lysate
from Cell
Suspension
Supernatant
from Cell
Suspension
Suspend cells in
20 mL PBS =
IPA-Killed Cell
Suspension
Figure 8 Flow chart for generation and analysis of IPA-killed cell suspensions. Data from analysis (blue boxes) is shown in Table 20
48
-------
Table 20 PCR Analysis of Components from Generation of IPA-Killed Cell Suspensions to Assess DNA Content or Loss
Sample
Type
PCR
Replicate
Ct by Component From IPA-Killed Cell Suspension Preparation*
Supernatant(#1)
Supernatant Wash
(#2)
Heat Lysate from Cell
Suspension
Heat Lysate Pellet
Supernatant
from Cell
Suspension
10-Fold
Dilution
50-Fold
Dilution
Undiluted
10-Fold
Dilution
Undiluted
10-Fold
Dilution
Undiluted
10-Fold
Dilution
Undiluted
IPA-
exposed
1
40.0
40.1
29.9
26.9
28.2
23.3
25.0
22.0
30.4
2
38.5
38.5
30.4
27.1
27.6
22.9
24.1
21.9
29.3
Ave (SD)
39.3 (1.1)
39.3 (1.1)
30.1 (0.3)
27.0 (0.2)
27.9 (0.4)
23.1 (0.2)
24.5 (0.6)
21.9 (0.1)
29.9 (0.8)
Control
1
31.2
28.8
32.2
29.7
29.8
25.7
26.0
23.6
34.5
2
31.5
28.9
32.8
29.7
28.7
25.5
25.0
23.5
33.2
Ave (SD)
31.4 (0.2)
28.9 (0.1)
32.5 (0.4)
29.7 (0.1)
29.2 (0.8)
25.6 (0.1)
25.5 (0.7)
23.6 (0.1)
33.8 (0.9)
* Cell suspensions were exposed to 70% isopropanol (IPA) for 2 lir. Ct data are from the YC2 assay using Universal reagents and Standard cycling conditions. Data
correspond to the blue boxes in Figure 8.
49
-------
RV-PCR experiments were conducted using IPA-killed Y pestis cells as background with 10 and
100 CFU/mL live cell concentrations. Two sets of experiments were conducted. The first
experiment included 102 and 104 dead cells/mL with 120 ± 30 or 12 ± 3 live cells (from reference
plating), while the second experiment used 105 and 106 dead cells/mL with 170 ± 20 or 17 ± 2 live
cells (from reference plating). In both cases, control treatments without dead cells were processed
in parallel. Aliquots were processed for DNA at To and T24 and analyzed using the YC2
chromosomal assay with both 10-fold and 100-fold dilutions to check for PCR inhibition and
ensure that the reaction was not saturated with DNA template from high levels of dead Y pestis
cells. Although the same data trends were evident, the 10-fold dilutions consistently showed more
positive detection compared with 100-fold dilution; the latter showing more non-detect results.
The potential issue of DNA saturation was not evident in these experiments so data from 10-fold
dilutions are shown.
Results from the first experiment for 10-fold diluted extracts showed that for the 100 live cell level
(for 10-fold diluted extracts), both dead cell backgrounds did not impact RV-PCR positive results
with average ACt values of 17.2 and 14.7 for 102 and 104 dead cells, respectively; these values
were similar to the 0 dead cell level (Ave ACt = 15.6; Table 21). For these treatments, 3 of 3
replicates were positive by RV-PCR. For the 10 live cell level with a 104 dead cell concentration
an average ACt of 6.1 was achieved; however, individual ACt values were 6.0, 7.9, and 4.5
showing that only 2 of 3 replicates met the criteria for positive detection. Positive PCR results
were obtained for the 102-dead cell concentrations with the average ACt of 15.6, while the control
without dead cells had an average ACt of 16.7. In Table 21, cases are highlighted where fewer
than 3 of 3 replicates were positive based on ACt> 6 (with the 24 hr incubation period) since these
would represent false negative results.
For the second experiment with higher levels of dead cells, the 102 live cell level still had average
ACt values > 6 for all dead cell backgrounds (for 10-fold diluted extracts), although for 106 dead
cells the individual ACt values were 7.1, 5.6, and 5.1 showing only 1 of 3 positive. It should be
noted however that these dead cell levels are quite high and would likely not be expected from
native water samples without some concentration method applied. For the 10 live cell level (10-
fold diluted extracts), the dead cell concentrations of 105 and 106 prevented positive detection,
since in all cases ACt values were < 6. For these high levels of dead cells, the control treatments
with 0 live cells showed negative results as expected with 0 of 3 positive; the data showed that the
method did not produce false positive results for high concentrations of dead cells.
These experiments served to bracket the conditions RV-PCR analysis could be used for detection
of live cells in post-decontamination scenarios. Based on these results, to improve the limit of
detection and reduce the false negative rate, a longer incubation could be warranted; an incubation
period of 36 hr could provide sufficient growth above the dead cell DNA background to detect live
cells at either the 10 or 100 live cell level. It is also recommended to analyze both undiluted and
10-fold diluted DNA extracts to minimize false positive/false negative rates.
50
-------
Table 21. RV-PCR Results for 10- and 100-Cell Levels (Live Y. pestis Cells) With Different
IPA-Killed Target Cell Concentrations - 10-Fold Diluted DNA Extracts
Sample Type
Ave CT (SD)*
Ave ACt
(SD)f
Positive
Replicates
Live Cell
Level
Dead Cell
Level
To
t24
100
0
41.8 (2.5)
26.2(1.0)
15.6 (3.9)
3 of 3
102
NDT
27.8 (3.3)
17.2 (3.8)
3 of 3
104
39.9(4.2)**
25.2 (0.7)
14.7 (4.9)
3 of 3
10s
34.1 (0.7)
26.3 (1.2)
7.8 (1.9)
3 of 3
106
31.3 (0.5)
25.4 (0.6)
6.0 (1.0)*
1 of 3
10
0
NDT
28.3 (0.7)
16.7 (0.9)
3 of 3
102
NDT
29.4 (0.6)
15.6 (0.7)
3 of 3
104
39.0 (2.7)
32.9 (2.8)
6.1 (1.7)t
2 of 3
10s
33.0 (1.1)
29.3 (0.9)
3.7 (1.3)
Oof 3
106
29.7 (0.2)
30.1 (0.4)
-0.4 (0.3)
Oof 3
0
0
NDT
NDT
0 (NA)
Oof 3
102
NDT
NDT
0 (NA)
Oof 3
104
38.2(1.8)
39.8 (1.1)
-1.4 (0.8)
Oof 3
10s
34.7(0.8)
34.8 (0.2)
-0.1 (1.0)
Oof 3
106
30.8 (0.3)
31.2 (0.4)
-0.4 (0.3)
Oof 3
* Average (Ave) and standard deviation (SD) based on triplicate PCR analyses.
** Average (SD) based on two replicates; NDT = Non-detect. Cases where less than 3 of 3 are positive based the
requirement that ACt > 6 are highlighted and delineated by a heavy border.
' Denotes case where average ACt value exceeds the criterion for positive detection, however
individual replicate ACt values were not all > 6.
51
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A replicate experiment was conducted using the conditions that produced data at the border
between 3 of 3 positive and < 3 of 3 positive, namely 104 - 106 dead cells with 100 live cells and
104 - 105 dead cells with 10 live cells. From reference plating, the actual live cells were 150 ± 30
for the 100 live cell level and 15 ± 3 for the 10 live cell level. The experiment was conducted as
that described previously and the results are shown in Table 22 for 10-fold DNA extracts. Based
on the results of the first two experiments where only 10-fold and 100-fold diluted DNA extracts
were analyzed, in this case undiluted extracts were also analyzed while the 100-fold dilutions were
not. The results from the replicate experiment were consistent with those shown in Table 21
however, in this case all replicates for the 105 dead cell level with 100 live cells were not positive
(although the average ACt > 6), showing ACt values of 7.5, 4.9, and 6.8. The replicate ACt values
for the 106 dead cell/100 live cell treatment were 11.0, 5.5, and 3.7. Forthe 10-cell level, the results
were consistent with the first replicate experiment except that the 104 dead cell/10 live cell
treatment showed 3 of 3 positive in this case.
52
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Table 22 RV-PCR Results for 10- and 100-Cell Levels (Live Y. pestis Cells) With Different
IPA-Killed Target Cell Concentrations - YC2 Assay With 10-Fold Diluted DNA Extracts
(Replicate Experiment)
Sample Type
Ave CT (SD) *
Ave ACt
(SD)
Positive
Replicates
Live Cell
Level
Dead Cell
Level
To
t24
100
0
NDT**
26.5 (1.6)
17.5 (0.4)**
2 of 2
104
37.2 (1.3)
26.7 (1.8)
10.5 (1.6)
3 of 3
10s
32.7 (2.2)
26.3 (1.1)
6.4 (1.4)t
2 of 3
106
31.8 (2.7)
25.1 (1.1)
6.7 (3.8) t
1 of 3
10
0
NDT
27.7 (0.7)
17.3 (0.8)
3 of 3
104
37.1 (0.8)
29.3 (1.0)
7.8 (1.0)
3 of 3
10s
33.2 (0.4)
28.3 (0.6)
4.9 (0.5)
Oof 3
0
0
NDT
NDT*
0.0 (NA)
Oof 3
104
35.9 (1.6)
36.4 (0.4)
-0.5 (2.1)**
Oof 2
10s
33.8 (0.7)
33.1 (0.7)
0.7 (0.4)
Oof 3
106
29.5 (0.7)
29.2 (0.1)
0.3 (0.7)
Oof 3
* Average (Ave) and standard deviation (SD) based on triplicate PCR analyses.
** Average (SD) based on two replicates; NDT = Non-detect. NDT set to 45 to calculate ACt.
Cases where less than 3 of 3 are positive based the requirement that ACt > 6 are highlighted and delineated by a heavy
border.
' Denotes case where average ACt value exceeds the criterion for positive detection, however
individual replicate ACt values were not all > 6.
The results for analysis of undiluted DNA extracts for the replicate experiment are shown in Table
23. The data between undiluted and 10-fold diluted extracts were similar as expected, although
undiluted extracts showed more positive results than 10-fold diluted extracts. It would be
advantageous to analyze both undiluted and 10-fold diluted extracts for critical samples—
53
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especially where decontamination or natural degradation may have contributed to high dead cell
concentrations—to minimize the false negative rate for this analysis. As mentioned the incubation
period could also be extended to 36 hr to ensure more accurate results. It should also be noted that
the negative controls with 0 live cells and high levels of dead cells did not show false positive
results (Table 23).
Table 23 RV-PCR Results for 10- and 100-Cell Levels (Live Y. pestis Cells) With Different
IPA-Killed Target Cell Concentrations - YC2 Assay With Undiluted DNA Extracts
(Replicate Experiment)
Sample Type
Ave CT (SD)*
Ave ACt
(SD)
Positive
Replicates
Live Cell
Level
Dead Cell
Level
To
t24
100
0
38.3 (1.8)
21.6 (0.6)
16.7(1.2)
3 of 3
104
33.4 (0.3)
22.5 (0.7)
10.9 (1.1)
3 of 3
10s
30.6 (0.9)
23.2 (0.6)
7.4 (0.8)
3 of 3
106
26.4 (0.3)
21.8 (0.9)
4.6 (0.8)
Oof 3
10
0
42.7 (3.3)*
24.3 (0.8)
18.4 (2.5)
3 of 3
104
33.8 (0.4)
26.3 (0.7)
7.5 (0.8)
3 of 3
10s
30.3 (0.5)
24.6 (0.6)
5.7 (0.5)
1 of 3
0
0
NDT
NDT**
0 (NA)
Oof 3
104
34.5 (0.3)**
33.5 (0.4)
1.0 (0.3)
Oof 3
10s
30.2 (0.6)
29.9 (0.5)
0.4 (0.6)
Oof 3
106
26.2 (0.9)
26.3 (0.5)
-0.1 (0.5)
Oof 3
* Average (Ave) and standard deviation (SD) based on triplicate PCR analyses.
** Average (SD) based on two replicates; NDT = Non-detect. NDT set to 45 to calculate ACt.
Cases where less than 3 of 3 are positive based the requirement that ACt > 6 are highlighted and delineated by a heavy
border.
54
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5.0 Conclusions
In this effort, an RV-PCR method for detection of viable Y pestis cells from water samples was
developed which also served as a model for vegetative cells of other pathogens. The RV-PCR
method was developed and optimized by improving procedures for high throughput culturing and
DNA extraction/purification. The overall method was then evaluated with regard to detection limit
and performance with complex water samples and high concentrations of dead target cells,
representing a range of possible real-world sample conditions.
Optimization of culturing procedures included use of YPEB in place of BHI broth for more
consistent growth in liquid culture. Experiments showed that 10X YPEB could be added to the
water sample to yield a IX broth concentration, thereby not significantly diluting the growth
medium. This broth consistently produced ~4-log cell growth over 24 hr for starting cell levels of
-101 -104 CFU/mL. In addition, TBA base (without blood) plates provided more reproducible Y
pestis colony growth compared with BHI agar plates to more accurately quantify resulting cell
suspensions.
Optimization of DNA extraction and purification protocols included streamlining the existing
Promega Magnesil kit procedure used for B. anthracis (Gram-positive) for the more readily-lysed
Y pestis (Gram-negative) cells; namely, two steps were removed (a lysis buffer wash and alcohol
wash step). The modification did not appear to have any negative effect on DNA yields obtained
since estimated CFU/mL from real-time PCR results were typically > 0.2-log higher compared
with CFU/mL from plate counts. Although PCR cannot be used to determine absolute CFU/mL
due to uncertainties in gene copy number per cell or per mass and variability in pipetting, PCR
efficiency, among others, the results suggested that cell lysis and DNA recovery were optimal for
this application.
The results of this effort indicated that a 24 hr incubation period was optimal for the 10-cell level
(10-99 cells) LOD for Y pestis. The same incubation period allowed maintenance of the 10-cell
level LOD even in the presence of high levels of insoluble and soluble potential chemical
interferences, high concentrations of live, non-target cells and spores, and high concentrations of
dead Y. pestis cells (102 - 104). However, for more complex samples or with >104 dead Y pestis
cell concentration (e.g., post-decontamination or clearance samples), the incubation time may be
extended to 36 hr to maintain the 10-cell level LOD. The RV-PCR method is expected to have an
advantage over traditional culture methods since isolated Y pestis colonies may be difficult to
detect in samples containing high concentration of non-target cells and spores.
The results from preliminary investigation into methods for cell concentration prior to RV-PCR
analysis (included modified filtration and immunomagnetic separation) did not provide an
advantage since the cell recovery was poor. In addition, the sample processing time was
significantly extended resulting in a longer time to results. Because of these results and the
perceived challenges for operational use, these cell concentration methods (as tested in this effort)
are not desirable for a rapid detection method. Other methods to concentrate cells prior to RV-
PCR analysis such as ultrafiltration could show better results in terms of LOD and reproducibility.
55
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Together these findings showed that the RV-PCR method could be readily applied to Y pestis in
water samples, demonstrating good sensitivity and method performance with complex sample
matrices. Though traditional culture methods are still the gold standard, the time to confirmed-
results can be 72 hr or more. In contrast, the RV-PCR method could provide results in less than
half this time. Compared to traditional culture methods, the RV-PCR method can also significantly
reduce the waste generated and the footprint for analysis. For example, just for growth, RV-PCR
uses a single 48-well plate for 48 samples (and controls) compared with the culture method that
uses 11 plates, dilution tubes, an enrichment culture tube, and additional plates for isolation and
subsequent confirmation by PCR.
Other existing manual or automated DNA extraction platforms can also be employed for RV-PCR
analysis. The advantage with the DNA extraction procedure used in this effort is that it minimizes
the use of centrifugation. The real-time PCR assays used in this effort consistently demonstrated
<10 genome equivalent LODs; however, other assays in use for detection of Y pestis could be
readily integrated into the RV-PCR method as well.
The RV-PCR method developed for Y pestis can be used as a model for optimizing methods
applicable to additional pathogens of concern including both bioterrorism threats and public health
threats. This rapid viability method enhances the capabilities of the ERLN to respond to bioattacks,
unintentional, or natural outbreak scenarios. More rapid results with the same or improved
accuracy compared to plating methods will aid decision makers in planning decontamination
efforts and determining if they are successful, thereby enabling safe, timely restoration and reuse.
56
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6.0 References
Amoako, K.K., M.J. Shields, N. Goji, C. Paquet, M.C. Thomas, T.W. Janzen, C.I.B. Kingombe,
A.J. Kell, and K.R. Hahn. 2012. Rapid detection and identification of Yersiniapestis from
food using immunomagnetic separation and pyrosequencing. Journal of Pathogens, 2012:
Article ID 781652, 6 pages, doi: 10.1155/2012/781652.
Buchrieser, C., C. Rusniok, L. Frangeul, E. Couve, A. Billault, F. Kunst, E. Carniel, and P. Glaser.
1999. The 102-kilobase pgm locus of Yersinia pestis: Sequence analysis and comparison
of selected regions among different Yersinia pestis and Yersinia pseudotuberculosis
strains. Infection and Immunity, 67(9):4851-61.
Derbise, A., V. Chenal-Francisque, C.leHuon, C. Fayolle, C. E. Demeure, B. Chane-Woon-Ming,
C. Me'digue, B. J. Hinnebusch, and E. Carniel. 2010. Delineation and analysis of
chromosomal regions specifying Yersinia pestis. Infection and Immunity, 78(9):3930-
3941.
Doran, T., Hanes, D., Weagant, S., Torosian, S., Burr, D., Yoshitomi, K., Jinneman, K., Penev,
R., Adeyemo, O., Williams-Hill, D., Morin, P. 2013. FERN Yersinia pestis Screening
Method, SOP No: FERN-MIC.0004.02, Issuing Authority: Food Emergency Response
Network (FERN).
US EPA, 2005. Method 415.3 Determination of Total Organic Carbon and Specific UV
Absorbance at 254 nm in Source Water. Revision 1.1. EPA/600/R-05/055.
US EPA Internal Report, 2010. Letant, S.E., V. Lao, E. Vitalis, M. Lam, S. Kane, T. Bunt, and S.
Shah. Development of Rapid Viability Polymerase Chain Reaction (RV-PCR) Protocols
for Yersinia pestis and Francisella tularensis. LLNL-TR-426322.
Gilbert, S.E., L.J. Rose, M. Howard, M.D. Bradley, S. Shah, E. Silvestri, F.W. Schaefer III, and J.
Noble-Wang. 2014. Evaluation of swabs and transport media for the recovery of Yersinia
pestis. Journal of Microbiological Methods, 96:35-41.
Hernandez, E., M. Girardet, F. Ramisse, D. Vidal, and J.-D. Cavallo. 2003. Antibiotic
susceptibilities of 94 isolates of Yersinia pestis to 24 antimicrobial agents. Journal of
Antimicrobial Chemotherapy, 52(6): 1029-31.
Himathongkham, S., M.L. Dodd, J.K. Yee, D.K. Lau, R.G. Bryant, A.S. Badoiu, H.K. Lau, L.S.
Guthertz, L. Crawford-Miksza, and M.A. Soliman. 2007. Recirculating immunomagnetic
57
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separation and optimal enrichment conditions for enhanced detection and recovery of low
levels of Escherichia coli 0157:H7 from fresh leafy produce and surface water. Journal of
Food Protection, 70(12):2717-24.
National Research Council (NRC). 1979. Iron. Baltimore, MD, University Park Press.
Rose, L.J., L. Hodges, H. O'Connell, and J. Noble-Wang. 2011. National validation study of a
cellulose sponge wipe-processing method for use after sampling Bacillus anthracis spores
from surfaces. Applied and Environmental Microbiology, 77(23):8355-8359.
US EPA. 2012. Protocol for Detection of Bacillus anthracis in Environmental Samples During the
Remediation Phase of an Anthrax Incident. Cincinnati, Ohio: U.S. Environmental
Protection Agency. EPA/ 600/R-12/577.
World Health Organization (WHO). 1996. Guidelines for Drinking-Water Quality, 2nd ed. Vol.
2. Health Criteria and Other Supporting Information. Geneva: World Health
Organization. WHO/SDE/WSH/03.04/08.
58
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Annex 1 Standard Operational Procedure -
LLNL Manual Protocol for Rapid Viability Polymerase
Chain Reaction (RV-PCR) for Analysis of Yersinia pestis in
Water Samples
DRAFT
Version 3.0
59
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Rapid Viability Polymerase Chain Reaction (RV-PCR)
This protocol describes processing and analysis of 40 mL water samples, first by stabilizing the
samples with addition of 4.5 mL of 10X PBS to bring the final PBS concentration to approximately
IX concentration. For RV-PCR analysis, the standard incubation period is 24 hr (to allow Yersinia
pestis cell propagation prior to DNA extraction and analysis); however, for post-decontamination,
field samples (with potentially high concentrations of dead Y pestis cells) the incubation period
may be extended to 36 hr to ensure that low concentrations of live cells can be detected in these
samples.
Acronyms
BSC Biosafety Cabinet
DI deionized
MOPs 3-(4-morpholino)propane sulfonic acid
PBS phosphate buffered saline
PMPs paramagnetic particles
PES polyethersulfone
RCF relative centrifugal force
Trademarked Products
Trademark
Holder
Location
AeraSeal™
Excel Scientific, Inc.
Victorville, CA
Bacto™
Difco Laboratories
Franklin Lakes, NJ
Biopur® Safe-Lock®
Eppendorf
Hamburg, Germany
Dynamag™
Life Technologies
Carlsbad, CA
MagneSil®
Promega
Madison, WI
MasterPure®
Epicentre Biotechnologies
Madison, WI
Millipore®, Milli-Q™
Millipore Corp.
Billerica, MA
TaqMan®
Life Technologies
Carlsbad, CA
Tyvek® suit
DuPont
Wilmington, DE
Ziploc®
Johnson and Johnson
New Brunswick, NJ
60
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Laboratory set-up
• Put PPE (personal protection equipment) on for Biosafety Level 3 (BSL-3): Tyvek®
suit, Powered Air Purifying Respirators (PAPR), booties, double gloves.
• Prepare fresh bleach solution (1 volume bleach + 9 volumes water). Date and label
with initials.
• Clean/bleach BioSafety Cabinet (BSC) and bench surfaces.
• All sample manipulations 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 rubber band(s)
• Squeeze bottle with 70% isopropyl alcohol
• Squeeze bottle with deionized (DI) water
• Autoclave tape
• Autoclave bags, aluminum foil, or Kraft paper
• Large photo-tray or similar tray for transport of racks
• Laboratory marker
• Timer
• Disposable aerosol filter pipettips: 1000 |iL, 200 |iL, 10 |iL (Rainin Cat. No. SR-
L1000F, SR-L200F, GP-10F or equivalent)
• 1.5 mL Eppendorf Snap-Cap Microcentrifuge Biopur® Safe-Lock® tubes (Fisher
Scientific Cat. No. 05-402-24B or equivalent)
• 50 mL conical tubes (VWR Cat. No. 21008-951 or equivalent)
• 15 mL conical tubes (VWR Cat. No. 21008-918 or equivalent)
• 250 mL and 1 L filter systems, polyethersulfone (PES), 0.2 |im (Fisher Scientific Cat.
No. 09-741-04, 09-741-03 or equivalent)
• Tubes, sterile 2 mL DNase, RNase-free, gasketed, screw caps (National Scientific
Cat. No. BC20NA-PS or equivalent)
• Glass Petri dishes, 100 x 15 mm
Supplies for RV-PCR Analysis
• Disposable nylon forceps (VWR Cat. No. 12576-933 or equivalent)
• 50 mL conical tubes (VWR Cat. No. 21008-951 or equivalent)
• Disposable serological pipets: 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 (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)
61
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• 2 mL Eppendorf tubes (Fisher Scientific Cat. No. 05-402-24C or equivalent)
• 96-well 2 mL tube rack (8 x 12 format) (Bel-Art Cat. No. 188450031)
• 48-well plates (E&K Scientific Cat. No. EK-2044 or equivalent)
Supplies for Real-time PCR Analysis
• 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)
• PCR-grade water
Supplies for Culture
• Petri dishes, sterile, disposable, 100 x 15 mm
• Inoculating loops and needles, sterile, disposable
• Disposable cell spreaders (such as L-shaped, Fisher Scientific Cat. No. 03-392-150 or
equivalent)
• Racks for 50 mL centrifuge tubes
• 50 mL conical tubes (VWR Cat. No. 21008-951 or equivalent)
• 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)
• Sterile, breathable adhesive seals (AeraSeal™ Film, Thomas Scientific Cat. No.
6980A25 or equivalent).
Equipment
• Biological Safety Cabinet (BSC) - Class II or Class III
• PCR preparation hood (optional)
• Shaker incubator for RV-PCR (Thermo Scientific, MaxQ™ 4000 Cat No. SHKE4000
or equivalent) and Universal 18" x 18" shaker platform (Thermo Scientific, MaxQ™
Cat. No. 30110)
• Balance, analytical, with Class S reference weights, capable of weighing 20 g ± 0.001
g
• ABI 7500 Fast Real-Time PCR System (Applied Biosystems Inc., Foster City, CA)
• Refrigerated centrifuge with PCR plate adapter and corresponding safety cups
(Eppendorf Cat. No. 5804R, 5810R or equivalent) or PCR plate spinner (placed in
BSC) (VWR, Cat. No. 89184-608 or equivalent)
• Refrigerated micro-centrifuge for Eppendorf tubes with aerosol-tight rotor
(Eppendorf, Cat. No. 5415R or equivalent)
62
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• Platform vortexer for RV-PCR (VWR Cat. No. 58816-115 or equivalent) with
Velcro® straps
• Single-tube vortexer
• Heating block for RV-PCR (VWR Cat. No. 12621-096 or equivalent) and 2 mL tube
blocks (VWR Cat. No. 12985-048 or equivalent) or water bath set at 95°C
• Single-channel micropipettors (1000 \\L, 200 |jL,100 |iL, 20 |iL, 10 |iL)
• Serological pipet aid
• Dynamag™ magnetic racks for RV-PCR (Invitrogen Cat. No. 123-21D or equivalent)
• Incubator(s), microbiological type, maintained at 28-30 °C
• Autoclave or steam sterilizer, capable of achieving 121°C (15 psi) for 30 minutes
• Cold block for 2 mL tubes (Eppendorf Cat. No. 3880 001.018 or equivalent)
Reagents
• PCR-grade water, sterile (Teknova Cat. No. W3350 or equivalent)
• Phosphate buffered saline (PBS) buffer (Teknova Cat. No. P0261 or equivalent)
• TE buffer (IX Tris-HCl-EDTA [Ethylenediaminetetraacetic acid]) buffer, pH 8.0,
Fisher Scientific, Cat. No. BP2473-500 or equivalent)
• Promega reagents for DNA extraction and purification procedure for RV-PCR:
• Magnesil® Blood Genomic, Max Yield System, Kit (Promega Cat. No. MD1360;
VWR Cat. No. PAMD1360)
• Salt Wash (VWR Cat. No. PAMD1401 or equivalent)
• Magnesil® Paramagnetic Particles (PMPs) (VWR Cat. No. PAMD1441 or
equivalent)
• Lysis Buffer (VWR, Cat. No. PAMD1392 or equivalent)
• Elution Buffer (VWR Cat. No. PAMD1421 or equivalent)
• Alcohol Wash, Blood (VWR Cat. No. PAMD1411 or equivalent)
• Anti-Foam Reagent (VWR Cat. No. PAMD1431 or equivalent)
• 100% Ethanol (200-proof) for preparation of 70% ethanol by dilution with PCR-
grade water
• TaqMan® Universal PCR Master Mix (Life Technologies, Cat. No. 4304437)
• Primers and probe for YC2 PCR assay targeting a hypothetical gene on the
chromosome of Y pestis
• Forward Primer (YP-EPA-YC2F) - 5 '-CAACGACTAGCCAGGCGAC-3'
• Reverse Primer (YP-EPA-YC2R) - 5'-CATTGTTCGCACGAAACGTAA -3'
• Probe (YP-EPA-YC2P) - 5'-6FAM-
TTTTATAACGATGCCT AC AACGGCTCTGC AA-BHQ1 -3'
• Primers and probe for YpMTl targeting a putative F1 operon positive regulatory
protein on the pMTl plasmid of Y pestis
• Forward Primer (YP-EPA-MTIF) - 5'-GGTAACAGATTCGTGGTTGAAGG-3'
• Reverse Primer (YP-EPA-MT1R) - 5' -CCCCACGGCAGTATAGGATG-3'
• Probe (YP-EPA-MT1P) - 5'-6FAM-
TCCCTTCTACCC AAC AAACCTTT AAAGGACC A-BHQ1 -3'
• Primers and probe for YpPl targeting thepla outer membrane protease gene on the
pPCPl plasmid of Y pestis
63
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• Forward Primer (YP -EPA-YP1F) - 5'-TGGGTTCGGGCACATGATA-3'
• Reverse Primer (YP -EPA-YP1R) - 5'-CCAGCGTTAATTACGGTACCATAA-
3'
• Probe (YP-EPA-YPIP) - 5'-6FAM-
CTT ACTTTCCGTGAGAAGAC ATCCGGCTC-BHQ1 -3'
Tryptose Blood Agar (TBA) plates (without blood)
1. Weigh 33 g Bacto™Tryptic Blood Agar Base powder into 1-L flask or bottle.
2. Add 500 mL MilliQ® H20.
3. Place mixture on hotplate and gently mix with spin bar.
4. Autoclave per manufacturer's directions.
5. Place on hotplate and gently mix with spin bar. Allow agar to cool down to 45°C
before pouring.
6. Pour 20 mL of solution in each petri dish using a serological pipette. Pouring is
performed in a sterile BSC.
IX Yersinia pestis Enrichment Broth (IX YPEB)
1. Weigh out the following:
25 g Bacto Heart Infusion Broth powder
6 g Yeast extract
3 g Soy tone
0.5 g Ferric Ammonium Sulfate
8.77 g MOPS buffering agent
2. Add 500 mL MilliQ H20.
3. Place mixture on stir plate and gently mix with spin bar.
4. Bring volume to 1000 mL.
5. Filter-sterilize using 1-L 0.22 |im cellulose acetate filtering system with
disposable bottle.
10X Yersinia pestis Enrichment Broth (10X YPEB)
1. Weigh out the following:
125 g Bacto Heart Infusion Broth powder
30 g Yeast extract
15 g Soy tone
2.5 g Ferric Ammonium Sulfate
43.85 g MOPS
2. Add 250 mL MilliQ H20.
3. Place mixture on stir plate and gently mix with spin bar.
4. Bring volume to 500 mL.
5. Filter-sterilize using 0.5-L or 1-L 0.22 |im cellulose acetate filtering system with
disposable bottle.
10% Bleach-pH amended (prepared daily)
• Prepare bleach solution by adding 1 part bleach, 1 part acetic acid and 8 parts
reagent-grade water as described below.
64
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• Add 2 parts water to 1 part bleach, then add 5% acetic acid (1 part) and remaining
water (6 parts). Measure pH and add bleach (to increase pH) or acetic acid (to
decrease pH) as needed to obtain a final pH between 6 and 7. A pH meter should
be used to measure pH as opposed to pH strips or kit. When mixed, place a lid on
the mixture to reduce chlorine escape and reduce worker exposure.
Sample Processing and Plating for Water Samples
Note: Gloves should be used and changed between samples and as indicated below.
1. Concentrate water sample by centrifugation
Note: if the water sample has not been previously stabilized by buffer addition to
maintain cell viability, add 4.5 mL of 10Xphosphate-buffered saline (PBS) to 40
mL water sample (final ~IXPBS concentration).
a. Using a 50-mL serological pipet, transfer 40 mL to a 50 mL screw capped
centrifuge tube. If the sample volume is greater than 40 mL, process the
sample by adjusting the PBS volume (final concentration ~1X PBS) and
the centrifugation step (Step lc., in multiple tubes, if necessary), to have a
final suspension volume of 3 mL (Step le.).
b. Repeat steps above for each sample.
c. Make sure tubes are balanced and place 50 mL tubes into sealing
centrifuge buckets and decontaminate centrifuge buckets before removing
them from the BSC.
d. Centrifuge tubes at 3,500 x g? with the brake off, for 15 minutes in a
swinging bucket rotor.
Note: A higher x g is preferred as long as the speed is within the tube specifications.
e. Remove the supernatant from each tube with a sterile 50 mL serological
pipet and discard leaving approximately 3 mL in each tube (or 3 mL total
if combining pellets from multiple tubes per sample). The pellet may be
easily disturbed and not visible, so keep the pipet tip away from the tube
bottom.
f. Remove suspension (or combined suspension) from one tube with a sterile
5 mL pipet (recording the volume) and transfer to well of 48-well plate.
2. Add concentrated growth medium and process for RV-PCR analysis.
a. Add 333 |iL of 10X YPEB to each well of the 48-well plate using a 1000
|iL pipettor (Final YPEB ~ IX). Mix well.
b. For each well, transfer 500 |iL from each well of the 48-well plate and
transfer to a screw cap tube. This is a To aliquot for each sample. Repeat
for each sample.
c. Store aliquots on ice or in cold block (4°C).
3. Seal and incubate 48-well plate
a. Seal 48-well plate with sterile, breathable seal.
b. Place in zip-lock bag and seal bag.
c. Incubate 48-well plate at 28°C on a shaker incubator at 180 rpm.
4. Process To aliquot for DNA extraction
a. Centrifuge tubes at 14,000 rpm (20,800 RCF) for 10 min at 4°C.
65
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b. Remove 300 |j,L supernatant and dispose to waste. Store pellets on ice or
in cold block (4°C). Alternatively, the pellet may be stored at -20°C until
ready to process for DNA extraction (see DNA Extraction/Purification
Procedure section).
5. At T24 (after 24 hr incubation), transfer 500 |iL from each well to a 2-mL screw
cap tube. This is a T24 aliquot for each sample.
Note: For post-decontamination, field samples (with potentially high
concentrations of dead Y. pestis cells), the incubation period may be extended to
36 hr.
a. Centrifuge tubes at 14,000 rpm (20,800 RCF) for 10 min at 4°C.
b. Remove 300 |j,L supernatant and dispose to waste. Store pellets on ice or
in cold block (4°C). Alternatively, the pellet may be stored at -20°C until
ready to process for DNA extraction.
6. Process the pellets from To and T24 aliquots by the DNA Extraction/Purification
Procedure below.
RV-PCR Analysis: DNA Extraction/Purification Procedure
Note: To and T24 extractions can be completed separately.
1. Thaw To and T24 aliquots if they were stored at -20°C.
2. Add 800 |iL of lysis buffer (VWR, Cat. No. PAMD1392 or equivalent) using a
1000 |iL pipettor with a new tip for each sample. Cap the tubes and mix by
vortexing on high (-1800 rpm) for 30 seconds and place in 96-well tube rack at
room temperature. Change gloves as necessary between samples.
3. Vortex each screw-cap tube briefly (low speed, 5-10 seconds) and transfer the
sample volume to a 2 mL Eppendorf tube (ensure the tubes are labeled correctly
during transfer). Change gloves as necessary between samples. Incubate the To
and T24 lysate tubes at room temperature for 5 minutes.
4. Vortex the paramagnetic particles (PMPs) on high (-1800 rpm) for 30-60
seconds, or until they are uniformly resuspended. Resuspend PMPs by briefly
vortexing (3-5 seconds) as necessary.
5. Uncap one tube at a time and add 600 |iL of PMPs to each To and T24 lysate
(containing 1 mL sample), hereafter referred to as "To and T24 tubes". Mix by
briefly vortexing (use a new tip for each sample and discard used tips in a sharps
container).
6. Repeat for all To and T24 tubes.
7. Vortex each To and T24 tube for 5 - 10 seconds (high), incubate at room
temperature for 5 minutes, briefly vortex, and then place on the magnetic stand
with hinged-side of the tube facing toward the magnet. After all the tubes are in
the stand, invert tubes 180 degrees (upside-down) turning away from you, then
right side-up, then upside down toward you, then right side-up (caps up) position.
This step allows all PMPs to contact the magnet. Check to see if any beads are in
the caps and if so, repeat the tube inversion cycle again. Let the tubes sit for 5 -
10 seconds before opening. Maintain the tube layout when transferring tubes
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between the magnetic stand and the 96-well tube rack. Alternatively, tubes may
be vortexed while in removable rack that interfaces with magnetic stand.
8. Uncap each tube, one at a time and withdraw all liquid using a 1000 |iL pipettor
with the pipet tip placed in the bottom of 2 mL tube, taking care not to disturb the
PMPs. Ensure that all the liquid is removed. Use a new pipet tip to remove any
residual liquid, if necessary. If liquid remains in the tube cap, remove by
pipetting. Dispose tip and liquid in a sharps container. Recap tube. Change
gloves as necessary.
9. Uncap each To and T24tube, one at a time, and add 360 |iL of lysis buffer using a
1000 |iL pipettor. Use a new tip for each sample and discard tips in a sharps
container. Cap and vortex on low setting for 5 - 10 seconds, and transfer to 96-
well tube rack.
10. After adding lysis buffer to all of the To and T24 tubes, vortex each tube for 5 - 10
seconds (low) and place back on the magnetic stand. After all tubes are in the
stand, follow tube inversion cycle, as described above.
11. Remove all the liquid as described above, except that a glove change between
samples is not required. Use a new tip for each To and T24 tube (discard used tips
in a sharps container). Recap the tube.
12. Repeat liquid transfer for all tubes.
13. 1st Salt Wash: Uncap each To and T24 tube, one at a time, and add 360 |iL of Salt
Wash solution (VWR Cat. No. PAMD1401 or equivalent). Use a new tip for each
To and T24 tube and discard used tips in a sharps container. Cap and transfer to
96-well tube rack.
14. After adding the Salt Wash solution to all of the To and T24 tubes, vortex each
tube for 5 - 10 seconds (low) and place on the magnetic stand. After all tubes are
in the stand, follow tube inversion cycle, as described above.
15. Remove liquid as described above. Use a new tip for each To and T24 tube and
discard used tips in a sharps container. Recap the tube. Repeat for all To and T24
tubes.
16. 2nd Salt Wash: Repeat Salt Wash for all To and T24 tubes.
17. 1st Alcohol Wash: Uncap each To and T24 tube, one at a time, and add 500 |iL of
alcohol wash solution (VWR Cat. No. PAMD1411 or equivalent). Use a new tip
for each sample and discard used tips in a sharps container. Cap and transfer to
96-well tube rack.
18. After adding alcohol wash solution to all of the To and T24 tubes, vortex each tube
for 5 - 10 seconds (low speed) and place on the magnetic stand. After all To and
T24 tubes are in the stand, follow the tube inversion cycle, as described above.
19. Remove liquid as described above. Use a new tip for each To and T24 tube and
discard used tips in a sharps container. Recap the tube.
20. 2nd Alcohol Wash: Repeat Alcohol Wash for all To and T24 tubes.
21. 3rd Alcohol Wash: Repeat Alcohol Wash for all To and T24 tubes except use 70%
ethanol wash solution. After the liquid is removed, recap the tube and transfer to
the 96-well tube rack.
22. Open all To and T24 tubes and air dry for 2 minutes.
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23. Heat the open To and T24 tubes in the heat block at 80°C until the PMPs are dry
(-20 minutes). Allow all the alcohol solution to evaporate since alcohol may
interfere with analysis.
24. DNA elution: While they are in the heating block add 200 |iL of elution buffer
(VWR Cat. No. PAMD1421 or equivalent) to each To and T24 tube, and close
tube.
25. Vortex for 10 seconds and let the tubes sit in the heating block for 80 seconds.
26. Briefly vortex the tubes (5-10 sec) taking care to prevent the liquid from
entering the tube cap and let the tube sit in the heating block for one minute.
27. Repeat vortexing/heating cycle four more times.
28. Remove the tubes from the heating block, place them in a 96-tube rack in the
BSC, and let them sit at room temperature for at least 5 minutes.
29. Briefly vortex each tube (5-10 seconds) on low speed. Place tube in 96-well
tube rack.
30. Briefly vortex each tube and place on the magnetic stand for at least 30 seconds.
Bring the cold block to the BSC.
31. Collect liquid from each To or T24 tube with a micropipettor (-80-90 |iL) and
transfer to a clean, labeled, 1.5 mL tube on a cold block (check tube labels to
ensure the correct order). Use a new tip for each tube and discard tips in a sharps
container. Visually verify absence of PMP carryover during final transfer. If
magnetic bead carryover occurred, place 1.5 mL tube on magnet, collect liquid,
and transfer to a clean, labeled, 1.5 mL tube (ensure the tubes are labeled
correctly during transfer).
32. If necessary, centrifuge tubes at 14,000 rpm at 4°C for 5 min to pellet any
particles remaining with the eluted DNA; carefully remove supernatant and
transfer to a new 1.5 mL tube using a new tip for each tube (ensure the tubes are
labeled correctly during transfer).
33. Store To and T24 DNA extract tubes "referred to as To and T24DNA extracts" at
4°C until PCR analysis (use photo-tray to transport 1.5 mL tubes in a rack).
Note: IfPCR cannot be performed within 24 hours. store DNA extracts at -20°C.
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.
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Real-time PCR Analysis
1. Prepare PCR Mix according to the table below (PCR Mix for All Selected Y. pestis
Assays).
2. Set up 96 well PCR plate with PCR mix according to plate layout in PCR-preparation
hood, seal, and transfer to BSC.
3. Analyze To and T24 DNA extracts on same PCR plate.
4. If samples were frozen, transfer them to BSC and let them thaw to room temperature.
5. Perform 1:10 dilution of samples. Alternatively, only run samples undiluted (5 |j,L plus
20 |j,L PCR Master Mix).
6. Add 90 |j,L of PCR-grade water to wells of a sterile 96-well plate. Note: 10-fold
dilutions may also be made in screw-cap tubes or 1.5 mL Eppendorf tubes.
7. Mix sample up and down 5 times and transfer 10 |j,L to plate wells, following the plate
layout.
8. Mix diluted samples up and down 10 times and transfer 5 |j,L from plate well or tube to
the PCR plate (with PCR Mix). Seal PCR plate with clear Edge Seal.
9. Centrifuge sealed PCR plate for 1 min at 2000 rpm.
10. Open safety cup in BSC, place plate on photo-tray, change gloves, transfer PCR plate to
ABI thermocycler.
11. Run PCR cycle (see below).
12. After cycle completion, discard sealed PCR plate to waste. Autoclave. PCR plates with
amplified product are never to be opened in the laboratory.
13. Follow laboratory cleanup procedure.
PCR Thermal Cycling Conditions
STEPS
l.Mi
incuhalion
AmpliTaq
(¦old
activation
PC U. 45 cycles
MOID
MOID
Denalii ration
A11 ilea ling/ex tension
Temperature
50°C
95°C
95°C
60°C
Time
2 min
10 min
5 sec
20 sec
Fast Ramp: 3.5°C/sec up and 3.5°C/sec down.
PCR Mix for All Selected Y. pestis Assays
l-'inal
Reagent
Volume (ill.)
Concentration
(MM)
TaqMan® 2X Universal Master Mix
12.5
IX
Forward primer, 10 |jM
0.5
0.20
Reverse primer, 10 |xM
0.5
0.20
Probe, 4 |iM
0.4
0.064
Molecular Biology Grade Water
6.1
N/A
Template DNA
5
Variable
TOTAL
25
69
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RV-PCR Data Interpretation
Calculate an average Ct from the replicate reactions for To and T24 DNA extracts of each sample.
Subtract the average Ct of the T24 DNA extract from the average Ct of the To DNA extract. If
there is no Ct value for the To or T24 DNA extract (i.e., non-detect), use 45 (total number of PCR
cycles used) as the Ct value. The significant change (decrease) in the average Ct value from To
to T24 (ACt) indicates a positive result suggesting the presence of viable Y pestis cells in the
sample. A ACt criterion of > 6 (an approximate two log difference in DNA concentration) and a
corresponding T24 Ct of < 39 was set. If an incubation time longer than 24 hours was used for the
RV-PCR, instead of T24, appropriate Tf (incubation time) should be used (i.e., 36 hr for post-
decontamination, field samples with high concentrations of dead Y pestis cells). However, (ACt)
> 6 algorithm should still be used for a positive result. 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 To Ct. A
minimum of two out of three T24 PCR replicates must result in Ct values < 39 to calculate the
average CTfor a sample result to be considered positive. Negative controls (No-Template Controls,
NTCs) should not yield any measurable Ct values above the background level. If Ct values are
obtained as a result of a possible contamination or cross-contamination, prepare fresh PCR Master
Mix and repeat analysis. In addition, field blank samples should not yield any measurable Ct
values. If Ct values are observed as a result of a possible contamination or cross-contamination,
a careful interpretation of the Ct values for the sample DNA extracts and field blanks must be done
to determine if the data is considered valid or if the PCR analyses must be repeated.
Traditional culturing of diluted cell suspensions on TBA (or other appropriate media)
1. Inoculate TBA plates with 100 |iL of each sample (each dilution is plated in triplicate).
2. Using one Lazy-L cell spreader per suspension, spread sample to obtain a uniform liquid
layer on plate.
Note: do not spread liquid to plate edge.
3. After all liquid is absorbed, invert plates.
4. Incubate TBA plates at 28°C for 3 days.
5. Place sealed sample tubes in a secondary container (re-sealable bag); store tubes at 4°C.
6. After 3 days, confirm growth for Y pestis. Confirm that a subset of the colonies is
characterized as Y pestis based on real-time PCR analysis using YC2 Y pestis-specific
chromosomal assay.
7. Count colony-forming units (CFU) and record on supplied data sheet:
• If colony counts are < 250 - record the actual number
• If colony counts are greater than 250, record as TNTC (too numerous to count)
70
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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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