EPA/600/R-19/240 | October 2019
www.epa.gov/homeland-security-research
United States
Environmental Protection
Agency
oEPA
Optimization of a Rapid Viability
Polymerase Chain Reaction (RV-
PCR) Protocol for Detection of
Francisella tularensis in Water
Samples
Office of Research and Development
Homeland Security Research Program
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EPA/600/R-19/240
October 2019
FINAL REPORT
Optimization of a Rapid Viability Polymerase Chain
Reaction (RV-PCR) Protocol for Detection of
Francisella tularensis in Water Samples
by
Sanjiv R. Shah, Ph.D.
U.S. Environmental Protection Agency
Cincinnati, OH 45268
and
Staci Kane, Ph.D.
Teneile Alfaro
Lawrence Livermore National Laboratory
United States Department of Energy
Livermore, CA 94551
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Disclaimer
U.S. Environmental Protection Agency
The United States Environmental Protection Agency (U.S. EPA) through its Office of Research
and Development funded and managed the research described here (EPA IA DW-89-92454101-
0). This report has been reviewed and approved for public release in accordance with the policies
of the U.S. EPA. Note that approval does not signify that the contents necessarily reflect the
views of the Agency. Mention of trade names, products, or services does not convey EPA
approval, endorsement, or recommendation.
Lawrence Livermore National Laboratory
This 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.
Disaster Characterization Branch
Homeland Security and Materials Management Division
Center for Environmental Solutions and Emergency Response
Office of Research and Development
U.S. Environmental Protection Agency
1300 Pennsylvania Avenue, NW
USEPA-8801RR
Washington, DC 20460
(202) 564-9522
shah, sani iv@epa. gov
If you have difficulty accessing these PDF documents, please contact
McCall.Amelia@epa.gov for assistance.
11
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Acknowledgments
Research Team
Staci Kane and Teneile Alfaro (Lawrence Livermore National Laboratory)
EPA Technical Lead
Sanjiv Shah [U.S. EPA Homeland Security & Materials Management Division
(HSMMD), Center for Environmental Solutions & Emergency Response
(CESER])
Technical Reviewers
Worth Calfee (U.S. EPA. HSMMD, CESER)
David Bright (U.S. EPA Office of Land and Emergency Management - Office of
Emergency Management - Consequence Management Advisory Division)
Latisha Mapp (U.S. EPA Office of Water - Water Security Division)
External Peer-Reviewers
Paul Morin (U.S. Food and Drug Administration)
Laura Rose (Centers for Disease Control and Prevention)
Quality Assurance Reviewer
Ramona Sherman (U.S. EPA. HSMMD, CESER)
Edit Reviewer
Marti Sinclair, (U.S. EPA. HSMMD, CESER: General Dynamics IT, EPA Contract
HHSN316201200050W)
iii
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List of Abbreviations and Acronyms
ABI Applied BioSystems, Inc.
ATD Arizona Test Dust
Avg average
B. anthracis Bacillus anthracis
BHI brain heart infusion
BHQ Black Hole Quencher
bp base pair
BSC biosafety cabinet
BSL-3 biosafety level-3
BVFH Brain Heart Infusion/Vitox/Fildes/Histidine
°C degree(s) Centigrade
CDC Centers for Disease Control and Prevention
CESER Center for Environmental Solutions and Emergency Response
CFU colony forming unit(s)
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 (T24) or T24 Ct Ct value after 24 hours incubation
Ct (T30) or T30 Ct Ct value after 30 hours incubation
Ct (T36) or T36 Ct Ct value after 36 hours incubation
ACt delta cycle threshold
DD distilled, deionized
DNA deoxyribonucleic acid
dsDNA double-stranded DNA
ERLN Environmental Response Laboratory Network
EPA U.S. Environmental Protection Agency
F. tularensis Francisella tularensis
FAM 6-carboxyfluorescein
fg/[iL femtogram(s) per microliter
g gram(s)
h hour(s)
HSMMD Homeland Security and Materials Management Division
HSRP Homeland Security Research Program
ISO International Organization for Standardization
kb kilobase
LLNL Lawrence Livermore National Laboratory
LRN Laboratory Response Network
Hg microgram(s)
Hg/L microgram(s) per liter
Hg/mL microgram(s) per milliliter
|j,m micrometer(s)
Mb mega base pairs
mg milligram(s)
iv
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min minute(s)
|j,L microliter(s)
mL milliliter(s)
mg/L milligram(s) per liter
MH2+ IX Mueller Hinton Broth 2 with supplements
mm millimeter(s)
mM millimolar
NA not applicable
NDT non-detect
ng/[xL nanogram(s) per microliter
NIST National Institute of Standards and Technology
nm nanometer(s)
NTC No-Template Control
OD600 optical density at 600 nm
ORD Office of Research and Development
PBS phosphate buffered saline
PCR polymerase chain reaction
pg picogram(s)
Plat Platinum
PMP paramagnetic particles
ppm part(s) per million
RCF relative centrifugal force
RNA ribonucleic acid
ROX 6-carboxyl-X-rhodamine
rpm revolution(s) per minute
RV rapid viability
RV-PCR rapid viability-polymerase chain reaction
SD standard deviation
sec second(s)
To time 0, prior to incubation
T24 after 24 h of incubation
T30 after 30 h of incubation
T36 after 36 h of incubation
Taq Taq DNA Polymerase (from Thermus aquaticus)
Tf time final, after incubation
TNTC too numerous to count
UNG uracil-N-glycosilase
VBNC viable but not culturable
WLA Water Laboratory Alliance
Y. pestis Yersinia pestis
YPEB Yersinia pestis Enrichment Broth
IX 1-fold concentrated (no concentration)
6X 6-fold concentrated
10X 10-fold concentrated
v
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Trademarked Products
Trademark
Holder
Location
ABI Gold™
Life Technologies
Carlsbad, CA
AB Applied BioSystems™
Life Technologies
Carlsbad, CA
AeraSeal™
Excel Scientific
Victorville, CA
AmpErase®
Thermo Fisher Scientific
Waltham, MA
AmpliTaq Gold®
Life Technologies
Carlsbad, CA
Bacto™
BD Biosciences
Franklin Lakes, NJ
BBL™
BD Biosciences
Franklin Lakes, NJ
BD BBL™
BD Biosciences
Franklin Lakes, NJ
Black Hole Quencher®
Biosearch Technologies
Petaluma, CA
Difco™
Becton, Dickinson & Co.
Franklin Lakes, NJ
Dynamag™
Life Technologies
Carlsbad, CA
Epicentre®
Epicentre Technologies Corp.
Madison, WI
Excel®
Microsoft® Corporation
Redmond, WA
Invitrogen®
Life Technologies
Carlsbad, CA
IsoVitaleX™
BD Biosciences
Franklin Lakes, NJ
Life Technologies™
Life Technologies
Carlsbad, CA
MagNA Pure®
Roche Diagnostics
Indianapolis, IN
MagneSil® Blood Genomic
Promega
Madison, WI
MasterPure®
Epicentre Technologies Corp.
Madison, WI
Millipore®, Milli-Q™
Millipore Corp.
Billerica, MA
PicoGreen®
Life Technologies
Carlsbad, CA
Platinum™ Taq Polymerase
Invitrogen
Carlsbad, CA
QIAamp®
Qiagen
Hilden, Germany
Quant-iT™
Life Technologies
Carlsbad, CA
Qubit®
Life Technologies
Carlsbad, CA
Remel™
Remel
Lenexa, KS
TaqMan®
Life Technologies
Carlsbad, CA
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Executive Summary
Francisella tularensis (!•'. tularensis), the pathogen that causes tularemia in humans and animals,
could be introduced into water infrastructure due to a natural outbreak, laboratory accident, or
intentional contamination, and it can remain viable and infectious for some time in water. It is a
category A select agent because it can readily be weaponized, has a low infective dose (1-10
cells), and can cause high morbidity and mortality. Due to these characteristics, national security
concerns have been raised regarding this biothreat agent.
Designated as the sector-specific agency for water and wastewater systems, the U.S.
Environmental Protection Agency (EPA) and, specifically, EPA's Office of Water would need
timely and reliable water sample analysis results during response to and recovery from a
waterborne tularemia outbreak. Regardless of the cause of contamination, there is a need for a
rapid and sensitive analytical method to detect viable F. tularensis.
A rapid viability polymerase chain reaction (RV-PCR) method could offer advantages over the
current plate culture-based method for viable F. tularensis detection, which requires incubation
in liquid or on solid media for 3-5 days followed by confirmation of culture growth or
presumptive pathogen colonies, respectively. For environmental samples with indigenous
microbes, culture analysis can be challenged by overgrowth of non-target microbes, which can
inhibit or mask growth from the slower-growing, fastidious F. tularensis cells. The RV-PCR
method combines short-incubation broth culture (relative to the plate culture-based method), and
sensitive, specific real-time polymerase chain reaction (PCR) analysis before and after sample
incubation to assess viability. The RV-PCR method 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 cells.
In a partnership between scientists at EPA's Homeland Security Research Program (HSRP)
within the Office of Research and Development (ORD) and those 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 viable F. tularensis detection. In particular, the method
was designed for use by the Water Laboratory Alliance, a network of laboratories established by
EPA's Office of Water for water sample analysis.
In this research effort, the RV-PCR method was developed and evaluated fori7, tularensis cells
with the following key features: 24-hour (h) to 36-h incubation (dependent on incident-specific
parameters), which was shortened from a 48-h incubation from previous efforts; sample
incubation in 48-well plates for high throughput culture and sample handling; 101-cell/sample
level (10-99 cells) sensitivity of detection; and a robust DNA extraction procedure that
accommodates complex sample backgrounds. By optimizing the cell processing procedure and
incorporating an automated DNA extraction/purification procedure (based on Roche MagNA
Pure® reagents), the method performance was further improved. Furthermore, the PCR assay
performance was significantly improved with addition of Platinum™ Taq Polymerase to the PCR
reagent mix. While the platforms and reagents evaluated here showed good DNA yield and
quality, other manual or automated platforms and associated reagents could be used for RV-PCR
analysis as well.
vii
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In this effort, previous work on !•'. tularensis growth optimization was leveraged and adapted for
development of concentrated growth medium. Further improvements in the broth culturing step
enabled reproducible growth even at low inoculum levels (10-20 cells per 3 mL water sample).
Addressing a range of potential "real world" complex sample types, additional recommendations
were made based on challenge testing with potential soluble and insoluble chemical
interferences, and with live, non-target or dead target biological interferences. Recommendations
included extending the incubation time to 36 hours to further reduce the method's false negative
rate for samples with low target cell numbers and high non-target microbial backgrounds.
While outside the scope of this effort, RV-PCR is expected to be compatible with ultrafiltration
for cell concentration since a 102-cell level (100-999 cells) method sensitivity was observed even
in complex samples containing chemical and biological interferences. The F. tularensis RV-PCR
method will help enhance the Water Laboratory Alliance capability for rapid, reliable, and high-
throughput water sample analysis in case of a man-made or natural tularemia outbreak.
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Table of Contents
Disclaimer ii
U.S. Environmental Protection Agency ii
Lawrence Livermore National Laboratory ii
Acknowledgments iii
List of Abbreviations and Acronyms iv
Trademarked Products vi
Executive Summary vii
List of Tables xi
List of Figures xii
1. Introduction 1
2. Materials and Methods 6
2.1 Bacterial Strains, Growth Media, and Incubation Conditions 6
2.2 Francisella tularensisSchu S4 Cell Cell Suspension Preparation 7
2.3 Sample Matrix Used in This Study 8
2.4 Addition of Dust Background 8
2.5 Preparation of FeSCU and Humic Acid Solutions as Challenge Materials 8
2.6 Rapid-Viability PCR Method 9
2.7 Automated DNA Extraction-Purification 10
2.8 Protocol for Francisella tularensis Schu S4 Inactivation for Automated DNA Extraction-
Purification in the Biosafety Level-3 (BSL-3) Laboratory 10
2.9 F. tularensis DNA Standards for Real-Time PCR 11
2.10 Real-Time PCR Analysis 11
2.11 Interpretation of RV-PCR Results 12
2.12 Data Analysis and Presentation 13
3. Quality Assurance and Quality Control 14
3.1 Laboratory Inspections 14
3.2 Calibration 14
3.3 Storage Conditions 14
3.4 Spiking 14
3.5 Real-time PCR Analysis 14
3.6 Replication 15
3.7 Controls 15
3.8 Data Quality Objectives/Data Quality Indicators 15
4. Results and Discussion 15
4.1 Task 1. Optimize F. tularensis growth in concentrated medium for RV-PCR analysis 16
4.1.1. Task Objectives. 16
4.1.2 Comparison and Selection of Growth Medium for F. tularensis Schu S4 Propagation 16
4.1.3 Evaluation ofF. tularensis Schu S4 Growth on 10X BVFH Diluted to IX Compared to IX
BVFH Medium 16
4.1.4 Evaluation ofF. tularensis Schu S4 Growth on 6X BVFH Medium Diluted to IX Compared to
IX BVFH Medium 17
4.1.5 Optimization ofF. tularensis Real-Time PCR Assays for RV-PCR Analysis 19
4.1.6. Evaluation ofF. tularensis Growth Schu S4 and RV-PCR Performance: Effect of Holding
Cells at 4°C Prior to Use 19
IX
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4.1.7Evaluation ofF. tularensis Growth Schu S4 and RV-PCR Performance Using 30- or36-H
Incubation 25
4.2 Task 2. Evaluate manual and automated DNA extraction-purification procedures for F.
tularensis 28
4.2.1 Objectives 28
4.2.2 Procedure for F. tularensis Schu S4 Inactivation for Automated DNA Extraction in the
BSL3 Laboratory 29
4.2.3 Evaluation of Manual and Automated DNA Extraction-Purification Procedures Using 103
and 104 F. tularensis Schu S4 CFU/mL 29
4.3 Task 3. Evaluate and optimize an RV-PCR protocol for F. tularensis Schu S4 detection in
water samples with relevant challenges 34
4.3.1 Objectives 34
4.3.2 Evaluation of chemical and biological challenges for RV-PCR analysis ofF. tularensis Schu
S4 in water samples 35
5.0 Summary and Conclusions 44
6.0 References 46
Appendix A. Standard Operating Procedure - Manual Protocol for Rapid Viability Polymerase
Chain Reaction (RV-PCR) for Analysis of Francisella tularensis in Water Samples 48
x
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List of Tables
Table 1. Nucleotide Sequences of the Primer/Probe Sets Used fori7, tularensis RV-PCR
Analysis 12
Table 2. Growth of F. tularensis Schu S4 on IX BVFH and BH2+ medium 16
Table 3. Growth of F. tularensis Schu S4 on Different Growth Media: 6X BVFH Diluted to IX
vs IX BVFH Medium 18
Table 4. Growth of F. tularensis Schu S4 on 6X BVFH Diluted to IX vs. IX BVFH Medium 18
Table 5. Real-time PCR Results for F4 and F5 assays fori7, tularensis 19
Table 6. RV-PCR and Culture Results fori7, tularensis Schu S4 Cells: Cells Used Without 4°C
Hold for 24 H 20
Table 7. RV-PCR and Culture Results for F. tularensis Schu S4: Cells Held for 24 H at 4°C. 21
Table 8. RV-PCR and Culture Results fori7, tularensis Schu S4: Cells Used Without 4°C 24-H
Hour Hold - Replicate Experiment
(Replicate Experiment) 23
Table 9. RV-PCR and Culture Results fori7, tularensis Schu S4: Cells
Held for 24 H at 4°C - Replicate Experiment 24
Table 10. Summary of RV-PCR and Culture Results (with 24-h Incubation) for Francisella
tularensis Cells With and Without a 24-Hour Hold at 4°C (Tables 6-9)* 25
Table 11. RV-PCR and Culture Results fori7, tularensis Schu S4 Cells Incubated for 30
Hours 26
Table 12. RV-PCR and Culture Results fori7, tularensis Schu S4 Cells Incubated for 36
Hours 26
Table 13. RV-PCR and Culture Results fori7, tularensis Schu S4 Cells Incubated
for 30 Hours - Replicate Experiment 27
Table 14. RV-PCR and Culture Results fori7, tularensis Schu S4 Cells Incubated for 36
Hours - Replicate Experiment 28
Table 15. Comparison of Promega and Roche DNA Extraction-Purification Procedures for
Detection of F. tularensis Cells Schu S4 by PCR After 30-Hour Incubation 31
Table 16. Comparison of Promega and Roche DNA Extraction-Purification Procedures for
Detection of F. tularensis Schu S4 Cells by PCR After 36-Hour Incubation 32
Table 17. Comparison of Promega and Roche DNA Extraction-Purification Procedures for
Detection of F. tularensis Schu S4 Cells by PCR After 30-Hour Incubation 33
Table 18. Comparison of Promega and Roche DNA Extraction-Purification Procedures for
Detection of F. tularensis Schu S4 Cells by PCR After 36-H Incubation 34
Table 19. RV-PCR Results fori7, tularensis Schu S4 Cells (-1,200 CFU/3-mL Sample) in the
Presence of Fe/Humics or ATD 36
Table 20. RV-PCR Results fori7, tularensis Schu S4 Cells (-120 CFU/3-mL Sample) in the
Presence of Fe/Humics or ATD 37
Table 21. RV-PCR Results fori7, tularensis Schu S4 Cells (-12 CFU/3-mL Sample) in the
Presence of Fe/Humics or ATD 38
Table 22. RV-PCR Results fori7, tularensis Schu S4 Cells (-2,500 CFU/3-mL Sample) in the
Presence of Fe/Humics or ATD - Replicate Experiment 40
Table 23. RV-PCR Results fori7, tularensis Schu S4 Cells (-250 CFU/3-mL Sample) in the
Presence of Fe/Humics or ATD - Replicate Experiment 41
XI
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Table 24. RV-PCR Results fori7, tularensis Schu S4 Cells (-25 CFU/3-mL Sample) in the
Presence of Fe/Humics or ATD - Replicate Experiment 42
Table 25. Summary of RV-PCR Results Based on Condition and Starting Cell Level 43
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 cells in the original sample. ... 3
Figure 2. Comparison of the RV-PCR method to the traditional culture method for
F. tularensis 3
Figure 3. Flow chart for RV-PCR analysis of F. tularensis cells from water samples 4
Xll
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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. The EPA is also designated
as the sector-specific agency for water and wastewater systems for this response phase. In this
role, EPA's decision makers will need timely and reliable water sample analysis results during
response to a microbial contamination incident and subsequent recovery efforts.
Francisella tularensis (/•'. tularensis), the pathogen that causes tularemia in humans and animals,
could be introduced into water infrastructure due to a natural outbreak, laboratory accident, or
intentional contamination. It is known that F. tularensis and several other vegetative bacterial
pathogens can remain viable and infectious for some time in certain environments including
water (Anda et al., 2001; Berrada et al., 2011). As reviewed by Rice (2015), drinking water
outbreaks of tularemia have been reported in the U.S. and several countries including Bulgaria,
Georgia, Germany, Italy, Kosovo, Norway, Russia, Sweden, and Turkey.
F. tularensis is a category A select agent because it can readily be weaponized, has a low
infective dose (1 - 10 cells; Jones et al., 2005; Saslaw et al., 1961), and high morbidity and
mortality. Therefore, national security concerns have been raised regarding this bacterium. As a
result, consensus-based recommendations have been developed for civilian defense if F.
tularensis is used as a biological weapon (Dennis et al., 2001).
F. tularensis is a Gram-negative coccobacillus whose main transmission vectors are arthropods
(ticks, deer flies), while small mammals (e.g., rabbits, muskrats) serve as reservoir hosts.
Protozoa have also been suggested as an important environmental reservoir (Abd et al., 2003). A
security concern surrounding this bacterium is its potential persistence in the environment. Due
to its historical usage as a biological weapon (Giircan, 2014) and the occurrence of natural
tularemia outbreaks, there is a need for rapid and sensitive analytical methods for detection of
viable F. tularensis in environmental samples.
Within EPA, the Office of Water is responsible for protecting and managing water resources.
The Office of Water 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), established as a national network of
laboratories. The WLA needs rapid and reliable sample analysis methods to assess the presence
of live F. tularensis. To help meet the need for more rapid and accurate detection of viable F.
tularensis in environmental samples, the EPA-HSRP, in collaboration with the Lawrence
Livermore National Laboratory (LLNL) of the Department of Energy, has developed and
optimized the rapid viability polymerase chain reaction (RV-PCR) method for rapid detection of
viable F. tularensis. This method as well as that developed for detection of viable Yersinia pestis
(which causes plague; US EPA, 2016) can serve as a template for the detection of other
vegetative bacterial pathogens, where modifications to the method can be made to accommodate
differences in growth requirements and characteristics.
1
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Methods to more rapidly determine pathogen presence and viability are needed as part of EPA's
capabilities to ensure public safety and to help mitigate impacts of facility and infrastructure
closures following a biological agent release. The current plate culture-based methods used for
detection of F. tularensis and other biothreat agents are labor-intensive and have a low
throughput (--30-40 samples processed per laboratory shift), with confirmed results obtained
only after several days. A review of methods for soil and water sample processing and analysis
fori7, tularensis described challenges with using traditional culture with these complex sample
types (US EPA, 2015). In fact, multiple instances were reported where culture identification was
unsuccessful.
While PCR methods typically have fewer deficiencies, it is well understood that rapid detection
methods such as real-time PCR cannot distinguish between live (potentially infectious) and dead
pathogens. However, features of real-time PCR were leveraged for development of RV-PCR,
which combines short-incubation broth culture (relative to the plate culture-based method) to
enable growth, with 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 characteristic of cell growth, referred to as the
change in cycle threshold (Ct) or ACt, between the initial (before sample incubation) Ct at time
0, pre-incubation (Ct To) and the Ct at time final, after incubation (Ct Tf). Example PCR
response curves are shown in Figure 1 along with the criteria for positive detection, namely,
ACt > 6 (which represents an increase in specific DNA content of ~2-log during incubation).
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 (as demonstrated for Bacillus
anthracis spores [Letant et al., 2011] and Y. pestis cells [S. Kane, personal communication]).
Furthermore, it is likely that viable but not culturable (VBNC) cells could be more readily
detected from liquid culture used in RV-PCR analysis compared with solid media used in
traditional plate culture analysis (Wai et al., 2000).
In addition, the RV-PCR method fori7, tularensis detection can provide a higher throughput
capability as compared to the traditional culture-based methods, and hence, can increase the
laboratory capacity for sample analysis. In place of multiple sample dilutions, solid agar plates,
and enrichment cultures per sample used by the culture method, the RV-PCR method uses a
single well per sample on a 48-well plate (Figure 2).
2
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700
GOO
500
d>
I too
o
(A
| 300
"" 200
100
0
Endpoint
response, /
aCt
ct
10
20 30
PCR Cycle
40
PCR analyses before (T„) 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
(To), Cr (Tf) and ACT
For a positive result,
ACT=(CT[T0]-Cr[Tf])>6
Figure 1. Example real-time PCR response curves showing parameters (Ct [To], Ct [Tf],
and ACt) used in determining presence or absence of viable cells 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) to
calculate a ACt value.
Plating Method
¦ 1 sample 11 culture plates + culture tube
presumptive F. tularensis colonies 2-5 PCR analyses/sample
• Confirmed results in - 72+ hr
Serial dilution and plating
Enrichment culturing
Filtration and plating
Figure 2. Comparison of the RV-PCR method to the traditional culture method for
F. tularensis.
3
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The effort reported here significantly expanded a previous effort where LLNL and EPA-ORD
scientists developed preliminary RV-PCR protocols fori7, tularensis and Y. pestis. The project
work, conducted under earlier effort, led to protocols for Y. pestis and F. tularensis cells from
wet wipes and buffered water samples (S. Letant, personal communication). 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 work reported
here. In addition, the EPA-sponsored work by Morris et al. (2017) to develop an optimal growth
medium for F. tularensis Type A strains was leveraged for this effort.
This project focused on detection of F. tularensis in a 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 microorganisms present in non-autoclaved ATD including Bacillus spp. and other non-
target bacteria as well as fungal species as identified by Rose et al. (2011). The virulent F.
tularensis Sehu S4 reference strain was used throughout, added at different inoculum levels per
water sample. In this effort, automated DNA extraction, concentration and purification steps
were incorporated to further shorten the timeline. Features of real-time PCR analysi s that benefit
rapid analysis include low detection limits (typically <10 DNA copies per reaction), several
order-of-magnitude concentration range (~8 logs), and the ability to detect low numbers of target
organisms in the presence of high populations of non-target organisms; whereas, traditional
culture methods are challenged with environmental backgrounds where target bacteria may be
outcompeted by indigenous microorganisms.
Water
Sample
Add 3 mL per
well; 48-well plate
Add 6X growth
medium
Mix; Take T0
aliquot for PCR
Incubate 37°C
24 - 30 h
Mix; Take Tf
aliquot for PCR
DNA extraction &
purification
T0 aliquot
PCR analysis
Viable cells
present, if
ACt>6
DNA extraction &
purification
Tf aliquot
PCR analysis
Figure 3. Flow chart for RV-PCR analysis of F. tularensis cells from water samples. Using a
48-well plate, 3.0 milliliter (mL) water sample was added with 0.6 mL 6X broth per 5-mL
well.
4
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The RV-PCR protocol steps and some of the equipment fori7, tularensis are shown in Figure 3.
In this study, manual DNA extraction was compared with automated extraction protocols to
enable higher throughput analysis, using the same robotic platform available in some laboratories
in the Centers for Disease Control and Prevention (CDC) Laboratory Response Network (LRN).
This report describes experiments and results focused on three major tasks:
• Task 1. Optimization of F. tularensis growth in concentrated medium for RV-PCR
analysis
• Task 2. Evaluation of manual and automated DNA extraction-purification
procedures for F. tularensis
• Task 3. Evaluation and optimization of an RV-PCR protocol for F. tularensis
detection in water samples with relevant challenges.
Tasks 1 and 2 included development and optimization of procedures fori7, tularensis 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: (1) humic acid and ferrous sulfate as potential chemical interferents, and (2) live
(native) ATD as a source of potential growth-competing microorganisms (non-target
cells/spores) and metal oxides.
As mentioned, previous growth medium development efforts (Morris et al., 2017) were
leveraged for Task 1. In this effort, different approaches to concentrate the growth medium were
evaluated to enable addition of a larger volume water sample, ultimately yielding a IX
concentration for RV-PCR analysis. A 10X concentrated growth medium was evaluated initially
based on successful development of a Y pestis RV-PCR method that used 0.3 mL 10X Y pestis
Enrichment Broth (US EPA, 2016; p. 12) with 2.7 mL water sample containing Y. pestis cells.
Based on initial poor results using 10X concentrated medium fori7, tularensis growth, a 6X
medium was also evaluated in this effort, showing improved results.
Previously, a low-throughput manual DNA extraction method with a modified Promega
MagneSil bead-based protocol was used (US EPA, 2016). In Task 2 of this effort, an automated
platform, Roche MagNA Pure® Compact instrument, was evaluated for inclusion in the F.
tularensis RV-PCR protocol. This platform as well as a higher throughput platform (e.g.,
MagNA Pure® LC) could be used to increase throughput for RV-PCR analysis. In addition,
automated processing would involve less handling of samples and therefore less risk of exposure
to the pathogen. Combined with automated DNA extraction, the RV-PCR protocol could further
shorten the timeline for detection of viable F. tularensis compared with traditional culture
approaches, or even enable detection from complex environmental samples where current
methods are limited (e.g., indigenous microbes could mask and/or inhibit growth of F.
tularensis). From previous efforts with B. anthracis Sterne cells, the Roche MagNA Pure®
Compact with associated extraction kits and protocols has been shown to provide good DNA
yields, with extracts showing on average 3.3 lower Ct values compared with the Promega
MagneSil® reagents (S. Kane, personal communication).
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This project focused on method optimization to detect the lowest possible number of viable F.
tularensis cells in water, targeting the 101-cell level (10-99 cells), using RV-PCR. In addition,
efforts to make the method more operational and to provide higher throughput capability were
conducted, followed by evaluation of the optimized method with reference challenge materials
relevant to water samples (Task 3). Chemical challenges included ferric sulfate and humic acid,
since these compounds have the potential to negatively affect cell growth, cell recovery from
water samples, and/or interfere with subsequent analysis (Schrader et al., 2012; Sidstedt et al.,
2015). In addition, microbial challenges included indigenous microorganisms present in native
reference dust (e.g., Arizona Test Dust ISO 12103-1; Powder Technology, Inc., Arden Hills,
MN). Controls were included to ensure that sample-processing protocols did not negatively
impact cell viability.
2. Materials and Methods
The following provides general information about the materials and methods used throughout the
study with more specific information provided with the individual experiments in the Results and
Discussion section.
2.1 Bacterial Strains, Growth Media, and Incubation Conditions
The virulent F. tularensis strain Schu S4 from the LLNL strain collection was used for all
experiments. The F. tularensis culture was grown in Brain Heart Infusion
(BHI)/Vitox/Fildes/Histidine (BVFH) medium or Mueller Hinton 2 medium (cation-adjusted)
with added glucose, ferric pyrophosphate, and IsoVitaleX™, referred to as IX Mueller Hinton
Broth 2 with supplements (MH2+) medium. The MH2+ medium was prepared as follows: (1) 11
g BBL™ Mueller Hinton Broth 2 (Cation-Adjusted) powder was added to 480 mL Milli-Q™ H2O,
which was mixed and heated to boiling; (2) the mixture was autoclaved at 121°C for 25 minutes;
(3) after allowing the broth to cool to room temperature, 5 mL 10% sterile glucose solution, 5
mL sterile 2.5% ferric pyrophosphate, and 10 mL sterile IsoVitaleX (BD BBL™ Biosciences,
Cat. No. 211876) were added aseptically; and (4) the medium was mixed and aliquoted into 50
mL tubes and refrigerated until use. The BVFH medium was prepared as described by Morris et
al. (2017) for a IX concentration. The IX BVFH medium contained: 3.7% weight (w)/volume
(v) Bacto™ BHI (BD Biosciences, Franklin Lakes, NJ, Cat. No. 237500); 0.1% (w/v) L-histidine
(Sigma-Aldrich, St. Louis, MO, Cat. No. H8000-100G); and 2% (v/v) Vitox Supplement (Oxoid,
Lenexa, KS; Cat. No. SR0090A); and 10% (v/v) Remel™ Fildes Enrichment (Remel, Lenexa,
KS; Cat. No. R45037).
For the RV-PCR method, use of concentrated growth medium was required so that a larger
volume water sample (up to 3 mL) could be analyzed. The 10X BVFH medium was prepared,
however, it was not possible to make one of the components, the Remel™ Fildes Enrichment, at a
10X concentration because it comes as a solution and is required at 10% in IX BVFH medium.
However, it was possible to accommodate the Fildes Enrichment at 5X. Briefly, the 10X BVFH
medium was prepared by (1) dissolving Bacto™ BHI powder and L-histidine into 30 mL
Millipore Milli-Q™ (Millipore Corp., Billerica, CA) purified water followed by autoclaving and
cooling the solution, (2) adding 20 mL of sterile Vitox Supplement, and (3) adding 50 mL of
sterile Fildes Enrichment. The 10X BVFH medium was subsequently diluted to a IX
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concentration with phosphate-buffered saline (PBS; Teknova, Hollister, CA; Cat. No. P0261), as
a surrogate for a water sample. As mentioned, the IX medium had a final concentration of 5%
Fildes.
Since it was observed that some medium components precipitated when the 10X BVFH medium
was prepared as described above, filter-sterilization (0.2 |im) was also evaluated. In this case, the
BHI powder and L-histidine were dissolved in 30 mL MilliQ® H2O and brought to boiling for 1
minute, (min) followed by cooling the solution. After cooling, 20 mL of sterile Vitox
Supplement and 50 mL of sterile Fildes Enrichment were added, and the solution was mixed.
The solution was then filter-sterilized with a disposable filtration system. The 10X solution was
stored at 4°C until use.
To optimize i7. tularensis growth, different diluents were also evaluated for dilution of 10X
BVFH to IX. The diluents included sterile PBS (pH 7.4), sterile Butterfield's buffer, or sterile
distilled, deionized (DD) H2O. The Butterfield's buffer was prepared by dissolving 34 g KH2PO4
in 500 mL Milli-Q™ H2O, adjusting the pH to 7.2, bringing the volume to 1 L with Milli-Q™
H2O, and autoclaving the solution for 15 min at 121°C. These media prepared with different
diluents all had a final 5% Fildes concentration after dilution to IX. For comparison, F.
tularensis growth was also evaluated in IX BVFH which either had a final concentration of 5%
or 10% Fildes Enrichment. Prior to autoclaving or filter-sterilizing, the pH was adjusted with
pellets of NaOH if the starting pH was below 7.
Later experiments used a 6X BVFH medium to address the issues of reduced Fildes Enrichment
concentration and the observed precipitation of medium components when the medium was
prepared at a 10X concentration. The 6X BVFH medium was prepared as follows: (1) BHI
powder and L-histidine were dissolved in 28 mL Millipore water and brought to boiling for 1
minute followed by cooling the solution, (2) 12 mL of sterile Vitox Supplement was added, and
(3) 60 mL of sterile Fildes Enrichment was added, and (4) the solution was filter-sterilized with a
disposable filtration system for a final concentration of 10% Fildes Enrichment when diluted to
IX. The 6X solution was stored at 4°C until use. There was no evidence of any insoluble
material or precipitate formed during storage of the 6X medium or during incubation/cell growth
for the 6X solution diluted to IX.
2.2 Francisella tularensis Schu S4 Cell Suspension Preparation
F. tularensis Schu S4 cell stocks were prepared in BVFH or MH2+ growth medium and grown
to mid-to late-log phase and then supplemented with 15% (v/v) glycerol for storage at -80°C.
Frozen stocks were used to start cultures on Chocolate Agar plates (Hardy Diagnostics, Santa
Maria, CA; Cat. No. E14) for experiments. Liquid growth medium was inoculated from cells on
3—day old streak plates. Initially, overnight 5-mL cultures were started from 2-3 colonies each
(from a streak plate) and were incubated with shaking (170 or 180 rpm) at 37°C. Later, to
provide more reproducible inocula, three selected colonies were suspended in 200 [xL or 500 [xL
PBS, which was mixed well, and 20 [xL were used to inoculate replicate 5-mL cultures. The
inoculum was also serially-diluted and plated to determine the number of cells added to the
culture. This 5-mL overnight culture was incubated for 15 to 24 hour (h), as specified below for
individual experiments. Typically, the overnight culture was prepared in IX BVFH and grown to
an OD600 (optical density at 600 nm) of - 1.5 which was diluted 10-fold in IX BVFH (~1 x
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109colony colony-forming units ([CFU)/]/mL), followed by centrifugation (3,000 x g for 10 min
at 4°C), washing one time in PBS buffer, and centrifugation was repeated. Then, the cell pellet
was suspended in sterile PBS buffer, and diluted in PBS to obtain the desired cell density.
In some cases, as specified below, the resulting culture suspension was then acclimated to low
nutrient conditions by storage in buffer at 4°C for 24 h prior to use (which also mimicked storage
at 4°C prior to sample analysis). In other cases, as described below, cells were used directly after
washing and resuspension, and compared to cells that were held for 24 h. The cells used directly
after washing and resuspension were also serially-diluted and plated to determine the number of
cells before storage at 4°C. The cells were also plated after the 24-h hold at 4°C to determine
any loss in cell viability.
2.3 Sample Matrix Used in This Study
As per EPA protocol (US EPA, 2017), a large volume water sample (1 - 2 L) is typically
collected and concentrated onto filter media, after which bacterial contaminants are recovered
from the filter by washing with PBS for subsequent analysis. Considering such use of PBS for
recovering and suspending the pathogens from the water samples, PBS (Teknova, Inc., Hollister,
CA; Cat. No. P0261) was used as a substitute for water samples because it maintained cell
viability and represented a reproducible matrix in terms of pH and chemical composition to
facilitate consistent experimental results during the RV-PCR method development. Throughout
the report, the term "sample" refers to /•'. tularensis Schu S4 cell suspensions prepared in PBS.
Materials were added to this buffer including: i (1) iron sulfate heptahydrate (Sigma-Aldrich,
Cat. No. 215422) and humic acid (Sigma-Aldrich, Cat. No. 53680-10G) to represent chemical
interferences and (2) ATD (Section 2.4) to represent chemical, biological (live, nontarget
microorganisms), and physical challenges.
2.4 Addition of Dust Background
ATD (International Organization for Standardization (ISO) 12103-1, A3 Medium Test Dust;
Powder Technology, Arden Hills, MN) was used to evaluate biological and chemical inhibition
effects on /•'. tularensis growth and PCR. Chemical composition analysis performed by the
manufacturer indicated the material consisted of: Si02 (68-76%), AI2O3 (10-15%), Fe203 (2-
5%),Na20 (2-4%), CaO (2-5%), MgO (1-2%), Ti02 (0.5-1.0%), and K20 (2-5%). Dust was
added at 4 mg/mL. While the microbial component is likely variable, the dust was shown to
contain ~5 x 104 CFU/10 mg background microbes including fungi and bacterial spores in
previous studies (Rose et al., 2011). Dust was nonsterilized, 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 hexahydrate and humic acid were added to samples to test for chemical interferences
affecting F. tularensis growth and PCR. Humic acid was used as a surrogate for natural organic
matter. An FeS04 solution was prepared in sterile distilled, deionized (DD) water and added to
samples at a final concentration of 10 micrograms (ng)/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 at the upper end of the range of values expected for drinking water samples
(NRC, 1979; WHO, 1996; US EPA, 2005).
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2.6 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. The method was like the method developed
for Y. pestis (US EPA, 2016), although 6X concentrated media were used. In contrast to the RV-
PCR protocol for B. anthracis spores (US EPA, 2017), multiple vacuum filtration steps for
concentration and buffer washes could not be used to concentrate vegetative cells and reliably
maintain their viability. Therefore, the water sample (PBS) was not vacuum filtered but rather
was prepared using 6X-concentrated broth, in the proper ratio, 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 3.0 mL water sample and 0.6 mL 6X broth were used.
After mixing by pipettor, a 0.5-mL aliquot removed from the total 3.6mL in each sample well
before incubation (time 0, prior to incubation (To) aliquot), transferred to 2-mL Eppendorf tubes,
and centrifuged at 20,800 relative centrifugal force (RCF) for 10 min at 4°C, after which 300 [j,L
were removed and discarded. The cell pellets in the remaining 0.2 mL were then frozen prior to
deoxyribonucleic acid (DNA) extraction and PCR analysis following the protocol detailed
below. During method development, in some cases (as specified in this report), 0.25 mL aliquots
were removed and processed as described above except that only 50 [xL was removed, leaving
0.2 mL pellets. The 48-well plate was sealed with a sterile AeraSeal™breathable adhesive seal
(Excel Scientific, Victorville, CA; Cat. No. BS-25) and incubated for different time periods from
18 to 40 h at 37°C with shaking at 180 revolutions per minute (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., three, eight-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 (re-suspended pellet) was processed for DNA extraction and
purification using the Promega paramagnetic particle (PMP)-based kit (MagneSil® Blood
Genomic, Max Yield System; Promega, Madison, WI; Cat. No. MD1360). This kit enables DNA
recovery from multiple complex samples simultaneously using a magnetic bead-based cleanup
method. The method used was the same as the method developed for Y pestis DNA extraction
(US EPA, 2016), which was modified from the method developed for B. anthracis cells (Letant
et al., 2011). When used with the appropriate buffers, the PMPs bind and later release DNA with
appropriate buffers resulting in DNA concentration and purification.
Briefly, the cell pellet in the remaining 200-|iL aliquot was thawed and 800-|jL Lysis Buffer
were added. The mixture was vortex mixed and incubated for 5 min. Next, 600-|jL of
paramagnetic particle (PMP) mix were added and mixed by vortexing. The tubes were placed on
a magnetic rack, 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
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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 cooled 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).
2.7 Automated DNA Extraction-Purification
The protocol for DNA extraction of F. tularensis cells on the automated Roche MagNA Pure
Compact instrument was based on the vendor's protocol using the MagNA Pure Compact
Nucleic Acid Isolation Kit I (Roche, Indianapolis, IN; Cat. No. 03730964001), but modified by
adding a heat lysis step, as described in section 2.8. The sample pellets taken at To and Tf were
thawed and treated by heat lysis at 70°C for 10 min prior to the Roche chemical lysis protocol.
After heat lysis, samples were cooled for three minutes and 300 |j,L Roche chemical lysis buffer
was added, followed by vortexing tubes for 10 seconds (sec) at 1,800-2,000 rpm, and incubation
at room temperature (22-25 °C) for 30 min. Samples were briefly vortexed every 5 min during
the 30-min incubation. Samples were processed on MagNA Pure® Compact robot with DNA-
Bacteria Purification Protocol (Appendix A).
2.8 Protocol for Frattcisella tularensis Schu S4 Inactivation for Automated DNA
Extraction-Purification in the Biosafety Level-3 (BSL-3) Laboratory
The protocol for inactivation of F. tularensis cells for subsequent DNA extraction on the
automated Roche MagNA Pure® Compact instrument was developed based on an LRN protocol
(CDC, personal communication). The protocol was required since F. tularensis cell lysates
needed to be removed from the biosafety cabinet (BSC) to complete the DNA extraction process
on the robot outside the BSC; this procedure constituted a removal from containment (per the
Centers for Disease Control and Prevention [CDC]) although the robot is in the same laboratory.
For approval of the inactivation procedure by the LLNL Biosafety Office, triplicates of both
inactivated samples and positive controls (not inactivated) were to be tested in three separate
experiments. In these tests, 100% of the inactivated material (or controls) were plated, with
plates incubated under optimal growth conditions for 96 h. Further guidance was provided from
CDC via the Select Agent Facility staff that an additional experiment was required to assess
whether the chemicals used to inactivate cells were sufficiently washed from the samples (by
repeated centrifugation and resuspension) prior to culture analysis. In this case, the inactivated
samples were split in half after wash steps, with half plated directly and the other half spiked
with a known amount of F. tularensis cells prior to plating (where growth would indicate
sufficient removal of the inactivating chemicals). After approval of the procedure, cell lysates
could be removed from the BSC and processed directly on the Roche platform.
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The inactivation procedure was reviewed by the Select Agent Facility staff including the LLNL
responsible official, and approved after some iteration. In addition, a checklist was generated to
document all the conditions of the experiments, including that starting cell densities were within
specified ranges and that all materials were within expiry dates. The checklist was also reviewed
and approved by the responsible official and Select Agent Facility staff for meeting adequate
biosafety procedures in the laboratory.
After reviewing preliminary data on cell inactivation with the initial chemical lysis (30 minutes
(min) at room temperature with mixing every 5 min), the EPA technical lead suggested adding a
step to perform heat lysis at 70°C for 10 min prior to conducting the Roche chemical lysis protocol.
Changes were made to the Inactivation Protocol and Checklist as appropriate, and the modified
procedure was submitted for review and approval by the LLNL responsible official.
2.9 F. tularensis DNA Standards for Real-Time PCR
F. tularensis DNA standards were generated from harvested cells from overnight incubation of 5
mL MH2+ cultures inoculated from 2-3 individual colonies from Chocolate Agar plates. A
MasterPure™ Complete DNA and RNA (ribonucleic acid) Purification Kit (Epicentre®
Technologies Corp., Madison, WI; Cat. No. MC85200) was used to extract genomic DNA from
the pure culture 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 many 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, Carlsbad, CA; Cat. No. Q32854) with a Qubit™ fluorometer (Cat. No. Q33216).
Standard concentrations prepared in PCR-grade water ranged from 1 femtogram (fg)/microliter
(|jL) to 1 nanogram (ng)/|jL. Each PCR plate contained seven 10-fold dilutions, ranging from 5
fg per 25-|jL PCR to 5 ng per 25-|jL 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 using computational tools for primer and TaqMan®
probe design, which were developed by Dr. Sanjiv Shah (while at Edgewood Chemical and
Biological Center of the U.S. Department of Defense) and LLNL, were used to design F.
tularensis real-time PCR assays (S. Letant, personal communication). In addition, in silico
analysis (i.e., performed via computer simulation) and rigorous wet-chemistry screening
approaches were used to further narrow the selection of candidate signatures, by screening
against an extensive panel of environmental extracts, bacteria, eukaryotes, near-neighbors, and
target strain DNAs. Furthermore, the selected assays were tested against target DNA templates to
yield sensitive assays. Two assays with the best sensitivity were selected for this effort to
optimize the RV-PCR protocol. Both assays target thepdpD, pathogenicity determinant protein
D unique to Type A F. tularensis with the F4 assay designed at LLNL and the F5 assay designed
at the Centers for Disease Control and Prevention (Kugeler et al., 2006).
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An Applied Biosystems® 7500 Fast Real-Time PCR System (Foster City, CA) was used to
perform real-time PCR. Each well of a 96-well PCR plate contained five-microliter (|jL) sample
aliquots added to 20 |j,L of PCR mix. The PCR mix (Appendix A, Page 64) contained TaqMan®
2X Universal PCR Master Mix (Life Technologies, Carlsbad, CA; Cat. No. 4304437), which
included AmpliTaq Gold® DNA polymerase, deoxynucleotide triphosphates, a 6-Carboxyl-X-
Rhodamine (ROX) passive reference dye (for signal normalization), and AmpErase® UNG
(uraciluracil-N-glycosilase), which prevented 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. Finally, Platinum™ Taq DNA Polymerase (Invitrogen, Cat. No.
10966026) was included at 0.25 |iL per reaction (1.25 U) since it was shown to improve assay
sensitivity. The assay primer and probe sequences are listed in Table 1. PCR-grade water
(Teknova, Cat. No. W3350) was used to make the mix volume up to 20 [xL per reaction, and 5
[j,L of sample was 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 per 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 Applied Biosystems, Inc. (ABI) Universal Master Mix was used
to normalize the fluorescent reporter signal. Automatic baseline and threshold settings were used
throughout.
Table 1. Nucleotide Sequences of the Primer/Probe Sets Used for F. tularensis RV-PCR
Analysis
Assay
Forward Primer*
Reverse Primer*
Probe*
Amplicon
Length
(bp)
F4
TTGCTCCAGTAGCTGC
AAGATT
CCAAGTGCTT
GGTGGTGGTA
FAM-
TGCTGCCGAGATGTT
TTCATTATTAACTGA
TGC-BHQ
125
F5
GAGACATCAATTAAAA
GAAGCAATACCTT
CCAAGAGTAC
TATTTCCGGTT
GGT
FAM-
AAAATT CTGCT C
AGCAGGATTTTGAT
TTGGTT-BHQ
105
* Sequences are listed in 5' to 3' orientation. Acronyms: Bp, base pair; FAM, fluorescein; BHQ, Black Hole Quencher
2.11 Interpretation of RV-PCR Results
As a starting point for RV-PCR detection of viable F. tularensis cells, the criteria developed for
B. anthracis and Y. pestis were employed. Specifically, for positive detection, the incubation
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) were required. For initial optimization, most of the work was
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conducted with 24-h or 30-h incubation, such that Tf = T24 or T30. For cases where no PCR
response was obtained (non-detect [NDT] results), the Ct values were set to 45 (since 45 PCR
cycles were used), to calculate ACt. A ACt > 6 represented an increase in DNA concentration of
approximately 2-log (100-fold) because 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 [1000-fold] in DNA concentration), and a
corresponding lower endpoint (Ct of < 36) could be used. For individual replicates within an
experiment, the RV-PCR result was considered positive when at least two of three replicates met
the algorithm requirement. Specifically, if two or three of three PCR replicates were NDT, then a
Ct value of 45 was assigned as the average To Ct (Ct value at time zero [pre-incubation]) or Tf
Ct (as appropriate) to calculate ACt; whereas, if two or three of three replicates showed positive
Ct values, the average of the positive Ct values was used to calculate ACt.
The RV-PCR method sensitivity of detection was equivalent to the F. tularensis cell level where
100% of the spiked samples had ACt > 6. This value was essentially an analytical sensitivity of
detection for the RV-PCR method and did not account for 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 time final after
incubation (Tf) Ct. Data tables show both individual PCR replicates as well as averages and
standard deviations calculated in Microsoft Excel®. As mentioned above, if a single PCR
replicate were positive and the other two replicates were non-detect, the sample was considered
NDT and the sample To or Tf Ct value was set to 45 to calculate ACt. Single replicate positive
high Ct values (e.g., 39-44) were likely due to cross contamination. For two or three positives of
three PCR replicates per sample, the average and standard deviation were calculated.
The overall SD from all sample replicates was calculated using the following equation,
Overall or joint SD = VjTfni — l)si2 + (n2 — 1 )s22 + (n3 — l)s32 + (711 x \X1 — X]2) +
(n2 x [X2 - X]2) + (n3 x [X3 - X]2)]/^ + n2 + n3 - 1)}
where nv n2, and n3 = the number of PCR analyses per sample for sample replicates 1, 2, and 3;
Sj, s2, and s3 = the standard deviation (SD) of the Ct values for the individual samples;
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.
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3. Quality Assurance and Quality Control
3.1 Laboratory Inspections
Monthly laboratory inspections were conducted by the project principal investigator to comply
with Department of Energy and 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 BSC, robotic enclosure, and
autoclave;
• Reviewing waste handling procedures;
• Taking inventory of select agents (in addition, 25% inventory conducted quarterly); and
• Reviewing personnel training.
3.2 Calibration
The ABI 7500 Fast PCR instrument was calibrated and underwent preventive 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 principal investigator.
3.3 Storage Conditions
An alarm system was used for refrigerators and freezers to ensure storage conditions were within
acceptable ranges. In addition, NIST-traceable temperature-recording devices were included
where PCR reagents and frozen cell 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 PBS) to test for cross-contamination was conducted for each experiment.
3.5 Real-time PCR Analysis
During the experiment, F. tularensis Schu S4 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 F.
tularensis Schu S4 cells as described in the Materials and Methods section.
14
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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 CFU for the spread plate method,
with corresponding 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
The objective of this research effort was to develop a qualitative, RV-PCR-based detection
method for F. tularensis. 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 the acceptable range (i.e., if a negative control showed one of three 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
one Ct value of the average). For individual replicates within an experiment, the RV-PCR result
was considered positive when at least two of three 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, specific details relevant to the given experiment are included
such as cell concentrations tested, broth used, and PCR assay employed, allowing the relevant
information to be near 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.
15
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4.1 Task 1. Optimize F. tularensis growth in concentrated medium for RV-PCR analysis
4.1.1. Task Objectives
The objective of the first task was to optimize growth of F. tularensis Schu S4 using
concentrated medium diluted to IX to analyze larger volume water samples under optimal
growth conditions. For high-throughput sample processing and analysis, the 48-well format was
used. This format was also used for RV-PCR analysis of Y. pestis, which served as the model for
this method. This task also included evaluation and optimization of real-time PCR assays and
assessment of the concentrated growth medium via RV-PCR analysis.
4.1.2 Comparison and Selection of Growth Medium for F. tularensis Schu S4 Propagation
In a preliminary experiment, IX BVFH medium was compared with IX MH2+ medium to
determine which produced higher Schu S4 cell densities determined spectrophotometrically. The
experiment was used to confirm results from an earlier study in which the BVFH medium was
shown to be superior to other growth media tested (Morris et al., 2017). MH2+ medium has been
used by researchers at LLNL to propagate F. tularensis. Similar but not identical media to MH2+
were tested by Morris et al. (2017). Overnight 5-mL cultures using both media types were started
from two colonies each (from a streak plate) and were incubated with shaking (170 rpm) at 37°C.
The OD600 values after 24-h incubation are shown in Table 2. The second experiment likely had
a slightly higher inoculum density since the final OD values were higher than the OD values for
the first experiment. For two different experiments, growth on BVFH produced higher OD
values compared to MH2+. Therefore, for subsequent method development, BVFH medium was
used.
Table 2. Growth of F. tularensis Schu S4 on IX BVFH and MH2+ medium
Growth
Medium
ODeoo after 24-h**
Expt. 1
Undiluted
Expt. 2
Undiluted
10-fold
Diluted
IX BVFH
1.26
>2*
0.31
MH2+
0.92
1.4
0.16
* The UV-Vis spectrometer plateaued at 2.
** OD6oo values are from duplicate measurements from a single aliquot (Expt. 1) or from single measurements
(Expt. 2). Dilution was performed using the same growth medium as for culturing cells.
4.1.3 Evaluation of F. tularensis Schu S4 Growth on 10X BVFH Diluted to IX Compared to
IX BVFH Medium
An experiment was conducted as described above although in this case, three F. tularensis Schu
S4 colonies were suspended in 200 |iL PBS, and then 20 |iL was used to inoculate replicate 5
mL cultures of either IX BVFH medium or 10X BVFH diluted to IX using PBS as diluent. This
inoculum preparation method was used for subsequent experiments as well. The starting cell
16
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density in this case was determined to be -2.7 x 107 CFU/mL. High numbers of cells were used
as inoculum so that UV-Vis spectroscopy could be used to estimate cell growth and screen
treatments for subsequent culture plating analysis. The 10X BVFH medium was prepared as
described in Section 2.1 (and in Appendix A). In this experiment, the pH of the 10X diluted
medium was not adjusted and when later tested, it was 6.62 ± 0.02 (for triplicate measurements)
and the pH for IX BVFH was measured at 7.16 ± 0.004. The lower pH could have negatively
impacted growth. When BHI was prepared as IX, the pH was -7.4. However, when prepared as
a 30 mL 10X stock, the pH was low (-6.6), and after autoclaving and adding the Fildes
Enrichment (50 mL) and Vitox Supplement (20 mL) the pH was 6.8.
The results from the growth experiment (15 h) showed that OD600 values for IX culture
replicates ranged from 0.5 to 0.35, while there was no change in OD values (i.e., no growth) for
the 10X diluted to IX culture (0.02). The OD measurements were confirmed by culture plating
showing - 2-log increase in growth on IX BVFH and no increase in cell number for 10X BVFH
diluted to IX (data not shown). While the final concentration of Fildes Enrichment is 10% in the
IX BVFH medium and 5% in the 10X BVFH medium diluted to IX, this factor was not thought
to account for the complete lack of growth with the latter medium. Alternatively, the poor F.
tularensis growth could have been due to the lower pH; therefore, a subsequent experiment was
conducted in which the 10X BVFH diluted to IX medium was pH-adjusted to 7.2 prior to
testing. In addition, other formulations were tested to trouble-shoot the poor growth on 10X
BVFH diluted to IX since we noted that some 10X BVFH medium components may have
precipitated, thereby effectively reducing the available (soluble) nutrient content. These other
formulations included Butterfield's Buffer or sterile ddH20 used as diluents instead of PBS.
Furthermore, 10X BVFH medium that was filter-sterilized (0.2-micron filter) was tested in
addition to one prepared by autoclaving the BHI/L-histidine stock prior to aseptic addition of
supplements. In all cases, the pH was adjusted with addition of NaOH pellets if the starting pH
was below 7. Additional information for media preparation is included in Section 2.1.
After medium preparation, 5-mL cultures were inoculated as described previously in this case, to
a final cell density of 8.1 x 106 CFU/mL from plating analysis. The 5-mL cultures were
incubated for 15 h at 37°C (180 rpm). In some cases, precipitates were observed for 10X medium
as particles adhered to the glass container. Whether any precipitates re-dissolved could not be
determined. However, this phenomenon was not ideal for standardized growth conditions, and
the media with and without inoculum (negative control) showed similar OD values. Only the IX
BVFH medium supported F. tularensis growth, with an ~2-log increase over the 15-h incubation
(data not shown). Based on these issues, a less concentrated medium was tested, that would still
enable use of a 3-mL water sample for a better sensitivity of detection, as described below.
4.1.4 Evaluation of F. tularensis Schu S4 Growth on 6X BVFH Medium Diluted to IX
Compared to IX BVFH Medium
Since the 10X BVFH medium diluted to IX did not support Schu S4 growth, a 6X growth
medium was tested (prepared as described in Section 2.1). This formulation also enabled a final
Fildes concentration of 10% when diluted to IX. PBS was used to dilute the 6X medium to IX,
and 5-mL cultures of each media type were inoculated as described (Section 2.2). Actual counts
were 1.5 x 107 CFU/mL from plating analysis. Schu S4 growth after 18.5 hours is shown in
17
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Table 3 as OD600 and CFU/mL from plating analysis. In addition, a p-value from a t-test
comparing CFU/mL for the two different media formulations is listed. Growth of Schu S4 on IX
BVFH medium was superior to the growth of Schu S4 on 6X diluted to IX (p = 0.01), although
both media types supported more than 2-log increase in cell number over the incubation period.
Table 3. Growth of F. tularensis Schu S4 on 6X BVFH Diluted to IX vs. IX BVFH Medium
Medium*
Sample
Replicate
OD600 after
18.5 h
Avg CFU/mL
after 18.5 h
t-Test (p-
value):
CFU/mL
1
1.28
7.97 x 109
6X BVFH
2
1.35
8.60 x 109
diluted to IX
3
1.31
7.97 x 109
Avg. (SD)*
1.31 (0.04)
8.18 (0.37) x 109
0.01
1
1.54
1.15 x 1010
IX BVFH
2
1.60
1.11 x 1010
3
1.47
1.13 x 1010
Avg. (SD)*
1.54 (0.07)
1.13 (0.022) x 1010
* Data are the average (Avg.) and standard deviation (SD) from triplicate samples.
A replicate experiment was conducted to compare the two media types. Five-mL cultures were
inoculated to yield starting cell numbers of 9.7 x 106 CFU/mL (from plate count analysis). After
incubation for 18.5 h, OD600 measurements were taken, and dilution series with plating was
conducted. For this experiment, the culture results were more comparable between media types
(Table 4). There was no significant difference between the 6X diluted to IX medium and the IX
medium (p ~ 0.18). Therefore, the 6X BVFH medium diluted to IX was used for the RV-PCR
method optimization.
Table 4. Growth of F. tularensis Schu S4 on 6X BVFH Diluted to IX vs. IX BVFH Medium
- Replicate Experiment
Medium
Sample
Replicate
OD600 after
18.5 h
Avg. CFU/mL
after 18.5 h
t-test (p-
value):
CFU/mL
6X BVFH
diluted to
IX
1
1.18
3.90 x 109
2
1.16
4.53 x 109
3
1.17
4.77 x 109
Avg. (SD)*
1.17(0.01)
4.40 (0.45) x 109
0.18
1
1.23
4.57 x 109
IX BVFH
2
1.28
7.43 x 109
3
1.21
5.60 x 109
Avg. (SD)*
1.24 (0.04)
5.87 (1.45) x 109
Data are the average (Avg.) and standard deviation (SD) from triplicate samples.
18
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4.1.5 Evaluation of F. tularensis Real-Time PCR Assays for RV-PCR Analysis
The F. tularensis F4 and F5 real-time PCR assays were tested with Schu S4 genomic DNA using
Universal 2X PCR Master Mix and standard conditions with and without inclusion of Platinum
(Plat) Taq DNA Polymerase (0.25 |iL per reaction) as described in Section 2.10. The genomic
DNA preparation measured by UV-Vis spectrometry showed 228 ng/|iL and the 260/280 ratio
was 2.05, showing good quality DNA. When measured by the Qubit HS dsDNA assay, the
quantity was 110 ng/|iL; the difference in quantities by methods was likely due to UV-Vis
spectroscopy measuring RNA in addition to DNA. Appropriate dilutions were made based on the
Qubit® measurement to test 500 picograms (pg) to 5 fg genomic DNA. The results are shown in
Table 5. The data showed that addition of Platinum™ Taq enzyme resulted in an average of 9.8
and 8.9 lower Ct values for the F4 and F5 assays, respectively. Therefore, Platinum™ Taq
polymerase was included in the PCR mix used for subsequent experiments.
Table 5. Real-time PCR Results for F4 and F5 assays for F. tularensis
DNA
(Pg)
Replicate
Avg. CT (SD)*
Ct
Difference
Avg. CT (SD)*
Ct
Difference
F4
F4 + Taq
F5
F5 + Taq
500
1
27.2 (0.4)
18.6 (0.2)
-8.6
27.6(0.6)
19.6(0.02)
-8.0
2
27.1 (0.6)
18.6 (0.1)
-8.5
27.3 (0.5)
19.4 (0.05)
-7.8
Avg. (SD)
27.2 (0.4)
18.6 (0.1)
-8.6 (0.1)
27.5 (0.4)
19.5 (0.09)
-7.9 (0.1)
50
1
30.7(0.6)
22.2 (0.07)
-8.5
30.8 (0.3)
23.0(0.09)
-7.8
2
30.9(0.7)
22.1 (0.1)
-8.8
31.2 (0.6)
22.9 (0.07)
-8.3
Avg. (SD)
30.8 (0.5)
22.2 (0.07)
-8.7 (0.2)
31.0 (0.4)
23.0 (0.07)
-8.1 (0.4)
5
1
35.1 (0.8)
25.7 (0.2)
-9.4
35.0(0.5)
26.4 (0.09)
-8.7
2
35.7(0.7)
25.7(0.09)
-10.0
35.3 (0.8)
26.3 (0.1)
-9.0
Avg. (SD)
35.4 (0.6)
25.7 (0.1)
-9.7 (0.4)
35.2 (0.5)
26.4 (0.08)
-8.9 (0.2)
0.5
1
39.8 (0.8)
29.2 (0.2)
-10.6
38.9(0.3)
30.0(0.06)
-9.0
2
40.1 (0.7)
29.2 (0.1)
-10.9
39.7(0.7)
29.2 (0.1)
-9.7
Avg. (SD)
40.0 (0.6)
29.2 (0.1)
-10.8 (0.2)
39.3 (0.5)
29.6 (0.4)
-9.4 (0.5)
0.05
1
43.8 (1.0)
33.0 (0.5)
-10.9
43.0(0.4)
33.2 (0.1)
-9.9
2
44.2 (0.9)
32.8 (0.2)
-11.4
44.1 (0.09)
33.5 (0.1)
-10.6
Avg. (SD)
44.0 (0.7)
32.9 (0.3)
-11.2 (0.4)
43.6 (0.5)
33.4 (0.2)
-10.3 (0.5)
0.005
1
NDT
36.3 (0.5)
NA
NDT
36.9 (0.3)
NA
2
NDT
36.3 (0.8)
NA
NDT
37.3 (1.2)
NA
Avg. (SD)
NA
36.3 (0.5)
NA
NA
37.1 (0.6)
NA
Overall Ct Difference Avg. (SD)
-9.8 (1.1)
-8.9 (1.0)
* Data are averages (Avg.) and standard deviations (SD) from three replicates. Data were generated using manual
threshold settings, and thresholds were the same for both assays with and without Plat Taq Polymerase (Taq).
NA, not applicable; NDT, non-detect.
4.1.6. Evaluation of F. tularensis Schu S4 Growth and RV-PCR Performance: Effect of
Holding Cells at 4°C Prior to Use
An experiment was conducted to determine whether 24 h was sufficient to detect viable cells
from water samples by RV-PCR using the established parameters including dilution of 6X
BVFH medium to IX with addition of the water sample (3 mL water sample plus 0.6 mL 6X
19
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medium) and incubation in 48-well plates (37°C, 180 rpm). Cells for inoculum were prepared as
described previously (Section 2.2). In addition, since cells may be held for up to 24 h prior to
sample receipt and analysis, a treatment was included where cells were held at 4°C for 24 h after
dilution in PBS to 104, 103, and 102 CFU per sample. This 4°C-hold treatment was compared
with cells prepared in PBS and used directly for culture and RV-PCR analysis. The starting
(inoculum) and ending (T24) cell densities were determined by dilution plating onto Chocolate
Agar, and 0.5 mL aliquots were analyzed at To and T24 by RV-PCR for both types of cells, those
used directly and those stored for 24 h.
Culture and RV-PCR results are shown in Table 6 for cells used directly and in Table 7 for cells
held for 24 h. The cells used directly were approximately 0.1 log higher (log CFU/mL) than
those that were held for 24 h. However, there was a greater log difference in growth when
incubated for 24 h at 37°C, -0.3 log higher for cells used directly on average. The log differences
ranged from -1.72 to 1.93 on average for cells used directly and 1.40 to 1.61 for cells held at 4°C
for 24 h.
Table 6. RV-PCR and Culture Results for F. tularensis Schu S4: Cells Used Without 4°C
Hold for 24 Hours
Starting
Cell
Density
(CFU/mL)
Sample
Replicate - PCR
Replicate
F5 Assay CT (SD)*
ACx
(T0-
T24)
T24 Cell
Density
Avg. CFU/mL
Cell
Density
Log
Change
To
T24
7.0 x 103
1 - 1
32.7
23.1
9.6
6.7 x 105
1.98
1-2
32.5
23.2
1-3
32.9
23.2
Avg. (SD)
32.7 (0.2)
23.1 (0.06)
2-1
32.1
23.7
8.3
7.2 x 105
2.01
2-2
31.8
23.7
2-3
32.1
23.8
Avg. (SD)
32.0 (0.2)
23.7 (0.05)
3 - 1
31.3
24.2
7.2
4.4 x 105
1.80
3-2
31.4
24.3
3-3
31.6
24.2
Avg. (SD)
31.4 (0.2)
24.2 (0.06)
Overall Avg.
NA
NA
8.4
6.1 (1.5) x 10s
1.93
7.0 x 102
1 - 1
35.2
27.3
7.6
3.3 x 104
1.67
1-2
35.4
27.3
1-3
34.2
27.3
Avg. (SD)
34.9 (0.6)
27.3 (0.04)
2-1
35.5
26.5
9.5
3.4 x 104
1.68
2-2
35.7
26.5
2-3
36.8
26.4
Avg. (SD)
36.0 (0.7)
26.5 (0.04)
3 - 1
35.0
27.2
7.6
4.6 x 104
1.82
3-2
35.0
27.4
3-3
34.7
27.4
20
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Avg. (SD)
34.9 (0.2)
27.3 (0.1)
Overall Avg.
NA
NA
8.2
3.7 (0.7) x 104
1.72
1 - 1
36.1
30.2
1-2
36.2
30.2
6.3
4.9 x 103
1.85
1-3
37.0
30.1
Avg. (SD)
36.5 (0.5)
30.2 (0.1)
2-1
39.2
30.7
2-2
NDT
30.8
8.0
4.7 x 103
1.82
7.0 x 101
2-3
38.1
30.7
Avg. (SD)
38.7 (0.8)
30.7 (0.07)
3 - 1
NDT
30.9
3-2
39.1
30.7
8.4
5.4 x 103
1.89
3-3
39.4
30.8
Avg. (SD)
39.2 (0.2)
30.8 (0.1)
Overall Avg.
NA
NA
7.6
5.0 (0.4) x 103
1.85
* Average (Avg.) and standard deviation (SD) for triplicate samples and triplicate PCR analyses per sample.
Acronyms: CFU, colony-forming units; Or. cycle threshold; NA, not applicable; NDT, non-detect.
Table 7. RV-PCR and Culture Results for F. tularensis Schu S4: Cells Held for 24 Hours at
4°C
Starting
Cell
Density
(CFU/mL)
Sample
Replicate - PCR
Replicate
F5 Assay CT (SD)*
ACt
(T0-
T24)
T24 Cell
Density
Avg. CFU/mL
Cell
Density
Log
Change
To
t24
5.5 x 103
1 - 1
33.4
25.3
7.9
1.5 x 105
1.42
1-2
33.6
25.7
1-3
33.2
25.5
Avg. (SD)
33.4 (0.2)
25.5 (0.2)
2- 1
32.1
25.6
6.5
1.7 x 105
1.41
2-2
31.8
25.4
2-3
32.3
25.7
Avg. (SD)
32.1 (0.3)
25.5 (0.1)
3- 1
33.0
25.5
7.1
1.4 x 105
1.35
3-2
32.7
25.6
3-3
32.3
25.7
Avg. (SD)
32.6 (0.4)
25.6 (0.07)
Overall Avg.
NA
NA
7.2
1.5 (0.2) x 10s
1.40 (0.04)
5.5 x 102
1 - 1
35.0
28.7
6.5
1.8 x 104
1.52
1-2
35.4
28.7
1-3
35.2
28.7
Avg. (SD)
35.2 (0.2)
28.7 (0.02)
2- 1
34.5
28.2
6.4
2.1 x 104
1.60
2-2
34.8
28.4
2-3
34.9
28.4
Avg. (SD)
34.7 (0.2)
28.3 (0.1)
3- 1
37.2
28.3
7.7
2.2 x 104
1.71
3-2
35.2
28.3
3-3
35.6
28.3
21
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Avg. (SD)
36.0 (1.1)
28.3 (0.01)
Overall Avg.
NA
NA
6.9
2.0 (0.2) x 104
1.61 (0.09)
1 - 1
37.3
32.7
1-2
NDT
32.5
5.2
1.8 x 103
1.61
1-3
38.2
32.3
Avg. (SD)
37.7 (0.7)
32.5 (0.2)
2- 1
NDT
31.9
2-2
NDT
32.2
12.9
1.9 x 103
1.45
5.5 x 101
2-3
38.2
32.2
Avg. (SD)
NDT
32.1 (0.2)
3- 1
38.4
32.6
3-2
38.4
32.1
5.7
1.3 x 103
1.53
3-3
37.4
32.3
Avg. (SD)
38.0 (0.6)
32.3 (0.2)
Overall Avg.
NA
NA
7.9
1.7 (0.3) x 103
1.53 (0.08)
* Average (Avg.) and standard deviation (SD) for triplicate samples and triplicate PCR analyses per sample.
Acronyms: CFU, colony-forming units; Ci. cycle threshold; NA, not applicable; NDT, non-detect.
These data suggested that the cells held for 24 h had a lag in growth compared to cells used
directly. Regardless, the ACt values were typically greater than 6 for individual replicates, for all
but two replicates at the lowest cell density after the 24 h, showing ACt values of 5.2 and 5.7
(the third replicate had ACt = 12.9). Individual ACt values ranged from 6.3 to 9.6 for cells used
directly and from 5.2 to 12.9 for cells used after being held for 24 h.
A replicate experiment was conducted as described with results shown in Table 8 for cells used
directly and Table 9 for cells used after holding at 4°C for 24 h. In this case, similar results were
obtained although the log differences from To to T24 were slightly higher for cells used directly
(compared to the first experiment), ranging from -1.86 to 2.13 on average, and ranging from
-1.46 to 1.69 on average for cells held at 4°C for 24 h. For this experiment, all had ACt > 6, with
individual ACt values ranging from 6.1 to 9.4 for cells used directly and from 6.4 to 7.7 for cells
used after being held for 24 h.
As a summary, the average log cell increase and ACt values (using 24-h incubation) for each
starting cell density and experiment are shown in Table 10. For the replicate experiments, the
average log CFU/mL increased -1.83-1.97 for cells used directly and -1.51-1.56 for cells held
first at 4°C for 24-h (Table 10). The corresponding average ACt values were -8.1 for cells used
directly and -6.6-7.1 for cells held first at 4°C for 24-h. Although the trend showed slightly
lower ACt values by holding cells for 24 h, there were no statistically significant differences (p >
0.01) between average ACt values for individual starting cell densities or for the average of all
three starting cell densities between treatments. However, for a few cases, the results were
significant for the average log CFU/mL increase (Table 10). Furthermore, those data suggested
that the RV-PCR method sensitivity of detection for viable F. tularensis cells using 24-h
incubation was in the range of 135 (starting cell density of 4.5 CFU/mL) to 210 (starting cell
density of 7.0 CFU/mL) cells per 3-mL water sample.
Improved method sensitivity would be expected with longer RV-PCR incubation periods.
Therefore, follow-on testing was performed using 30- and/or 36-h incubation periods. The RV-
PCR data from DNA extracts used the manual Promega protocol since the automated Roche
22
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MagNA Pure® protocol was not yet approved for use in the Biosafety Level biosafety level-3
(BSL-3) laboratory. Based on results from comparison studies between manual and automated
DNA extraction/purification protocols for Bacillus cmthracis (B. cmthracis), we thought that
lower Ct values would be generated using the automated platform. However, in this case, the
ACt values could be similar if lower Ct values are noted for both To and T24 (or later) time
points.
Table 8. RV-PCR and Culture Results for F. tularensis Schu S4: Cells Used Without 4°C
24-Hours Hold - Replicate Experiment
Starting
Cell Density
(CFU/mL)
Sample Replicate -
PCR Replicate
F5 Assay Ct (SD)*
ACt (To-
T24)
T24 Cell
Density
(CFU/mL)
Cell Density
Log Change
To
T24
4.5 x 103
1 - 1
31.0
21.7
9.4
8.3 x 105
2.26
1-2
31.2
21.9
1-3
31.4
21.8
Avg. (SD)
31.2 (0.2)
21.8 (0.09)
2-1
31.7
24.1
7.8
nd**
ND**
2-2
31.8
24.1
2-3
32.0
23.9
Avg. (SD)
31.8 (0.2)
24.1 (0.1)
3 - 1
31.6
22.3
9.1
4.0 x 105
1.94
3-2
31.2
22.3
3-3
31.4
22.4
Avg. (SD)
31.4 (0.2)
22.3 (0.04)
Overall Avg. (SD)
NA
NA
8.7 (0.9)
6.1 (2.2) x 10s
2.13 (0.23)
4.5 x 102
1 - 1
34.6
26.4
8.2
3.9 x 104
1.94
1-2
35.0
26.6
1-3
34.6
26.4
Avg. (SD)
34.7 (0.2)
26.5 (0.09)
2-1
35.1
26.5
8.3
4.0 x 104
1.94
2-2
34.7
26.5
2-3
34.5
26.5
Avg. (SD)
34.8 (0.3)
26.5 (0.02)
3 - 1
35.0
26.7
8.2
3.4 x 104
1.87
3-2
34.6
26.8
3-3
35.7
27.0
Avg. (SD)
35.1 (0.6)
26.9 (0.2)
Overall Avg. (SD)
NA
NA
8.2 (0.03)
3.8 (0.9) x 104
1.92 (0.04)
4.5 x 101
1 - 1
38.7
29.9
8.1
3.7 x 103
1.91
1-2
38.4
29.9
1-3
37.3
30.2
Avg. (SD)
38.1 (0.7)
30.0 (0.2)
2-1
NDT
30.2
7.9
2.9 x 103
1.81
2-2
38.2
30.3
2-3
38.3
30.5
Avg. (SD)
38.2 (0.1)
30.3 (0.2)
3 - 1
36.6
30.3
6.1
3.2 x 103
1.85
3-2
36.2
30.4
23
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3-3
NDT
30.2
Avg. (SD)
36.4 (0.3)
30.3 (0.06)
Overall Avg. (SD)
NA
NA
7.4 (1.1)
3.3 (0.5) x 103
1.86 (0.05)
* Average (Avg.) and standard deviation (SD) for triplicate samples and triplicate PCR analyses per sample.
** ND = Not determined. Sample replicate had contamination with non-/<. tularensis colonies such that accurate
Schu S4 colony counts could not be obtained. Acronyms: CFU, colony-fonning units; Ci. cycle threshold; NA, not
applicable; NDT, non-detect.
Table 9. RV-PCR and Culture Results for F. tularensis Schu S4: Cells Held for 24 Hours at
4°C - Replicate Experiment
Starting
F5 Assay Ct*
T24 Cell
Cell
Density
(CFU/mL)
Sample Replicate
- PCR Replicate
To
t24
ACt (To-
t24)
Density
Avg.
CFU/mL
Cell Density
Log Change
1 - 1
31.2
23.4
1-2
31.0
23.5
7.6
2.6 x io5
1.75
1-3
31.1
23.5
Avg. (SD)
31.1 (0.07)
23.5 (0.05)
2-1
31.3
23.5
2-2
31.3
23.5
2-3
31.2
23.6
7.7
2.9 x 105
1.80
4.6 x 103
Avg. (SD)
31.3
(0.006)
23.5 (0.05)
3-1
31.2
24.0
3-2
31.2
23.8
7.3
1.5 x 105
1.52
3-3
31.2
23.7
Avg. (SD)
31.2 (0.03)
23.9 (0.1)
Overall Avg (SD)
NA
NA
7.6 (0.2)
2.3 (0.7) x
10s
1.69 (0.15)
1 - 1
35.4
27.8
1-2
34.8
27.9
7.2
1.3 x 104
1.44
1-3
34.7
27.7
Avg. (SD)
35.0 (0.4)
27.8 (0.09)
2-1
33.5
27.4
2-2
34.5
27.7
6.2
1.8 x 104
1.59
4.6 x 102
2-3
33.5
27.6
Avg. (SD)
33.8 (0.5)
27.6 (0.2)
3-1
34.8
27.4
3-2
34.4
27.5
7.1
1.7 x 104
1.56
3-3
34.6
27.6
Avg. (SD)
34.6 (0.2)
27.5 (0.1)
Overall Avg. (SD)
NA
NA
6.8 (0.5)
1.6 (1.0) x
104
1.53 (0.08)
1 - 1
NDT
31.0
1-2
NDT
30.9
7.2
1.5 x 103
1.51
1-3
38.2
30.9
4.6 x 101
Avg. (SD)
38.2
30.9 (0.01)
2-1
37.4
30.9
2-2
NDT
31.1
7.4
1.4 x 103
1.48
2-3
39.7
31.3
24
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Avg. (SD)
38.5 (1.6)
31.1 (0.2)
3-1
38.9
32.1
6.4
1.2 x 103
1.40
3-2
38.6
32.0
3-3
37.5
31.7
Avg. (SD)
38.3 (0.7)
32.0 (0.2)
Overall Avg. (SD)
NA
NA
7.0 (0.5)
1.3 (0.2) x
103
1.46 (0.06)
* Average (Avg.) and standard deviation (SD) for triplicate samples and triplicate PCR analyses per sample.
Acronyms: CFU, colony-forming units; Or. cycle threshold; NA, not applicable; NDT, non-detect.
Table 10. Summary of RV-PCR and Culture Results (with 24-h Incubation) for
F. tularensis Schu S4 Cells With and Without a 24-Hour Hold at 4°C (Tables 6-9)*
Starting Cell
Density
Avg. (SD)** Log CFU/mL
Increase (To - Tm)
Avg. (SD)** ACt
(To-T24)
No 24-H Hold
24-H Hold
No 24-H Hold
24-H Hold
Experiment #1
101
1.85 (0.03)
1.53 (0.08)
7.6(1.1)
5.7(0.4)
102
1.72 (0.08)
1.61 (0.09)
8.2 (1.1)
6.9(0.7)
103
1.93 (0.12V
1.40 (0.04)
8.4 (1.2)
7.2 (0.7)
Experiment #2
101
1.86 (0.05)t
1.46 (0.06)
7.4 (1.1)
7.0(0.5)
102
1.92 (0.04)
1.53 (0.08)
8.2 (0.03)
6.8 (0.5)
103
2.13 (0.23)
1.69 (0.15)
8.7 (0.9)
7.6(0.2)
* The average log cell increase and ACt values from Tables 6-9 are summarized here.
**Average (Avg.) and standard deviation (SD) were calculated from triplicate samples and triplicate analyses per
sample. The SD was calculated using the equation in Section 2.12.
Denotes significantly greater value (p <0.01; paired, two-tailed T-test) for comparisons between with and without
4°C hold for different starting cell densities or for all three cell densities (lO'-lO3 CFU/mL).
Acronyms: CFU, colony-fonning units; Ct. cycle threshold; H, hour.
4.1.7 Evaluation of F. tularensis Schu S4 Growth and RV-PCR Performance Using 30- or 36-
h Incubation
From the RV-PCR experiment using 24-h incubation, with cells held for 24 h at 4°C prior to use,
the lowest cell level (-165 CFU/3-mL water sample) showed 2 of 3 replicates with a ACt value
of < 6 (5.2 and 5.7; the 3rd replicate had ACt= 6.1); therefore, we tested both 30-h and 36-h
incubation to determine if the ACt cut-off for positive detection could be achieved for all sample
replicates. This test was important since more complex samples containing growth and/or PCR
inhibitors could have even lower ACt values. Like the previous experiment, 6X BVFH medium
was diluted to IX with addition of the water sample (3 mL PBS with Schu S4 cells plus 0.6 mL
6X medium), and the inoculum was prepared as described previously. Two separate 48-well
plates were used for the two different incubation periods.
25
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Table 11. RV-PCR and Culture Results for F. tularensis Schu S4 Cells Incubated for 30
Hours
Starting Cell
Density
(CFU/mL)
Sample
Replicate*
ACt (To—T30)
T30 Cell Density
Avg. CFU/mL
Cell Density
Log Change
1
11.9
2.1 x 106
4.1 x 103
2
10.9
2.0 x 106
2.83
3
10.9
1.4 x 106
Avg. (SD)
11.2 (0.5)
1.81 (0.4) x 106
1
9.7
1.3 x 105
4.1 x 102
2
11.9
1.4 x 105
2.54
3
9.5
1.6 x 105
Avg. (SD)
10.4 (1.3)
1.44 (0.14) x 10s
1
13.3
1.3 x 104
4.1 x 101
2
12.4
7.8 x 103
2.73
3
12.3
8.3 x 103
Avg. (SD)
12.7 (0.6)
9.54 (2.60) x 103
1
9.5
1.2 x 103
4.1
2
8.3
9.0 x 102
2.83
3
9.8
1.4 x 103
Avg. (SD)
9.2 (0.8)
1.19 (0.27) x 103
* Average (Avg.) and standard deviation (SD) for triplicate samples. Data are from manual DNA extraction.
Acronyms: CFU, colony-forming units; Or. cycle threshold.
Table 12. RV-PCR and Culture Results for F. tularensis Schu S4 Cells Incubated for 36
Hours
Starting Cell
Density
(CFU/mL)
Sample
Replicate*
ACx (T^T36)
T36 Cell Density
Avg. CFU/mL
Cell Density
Log Change
1
16.0
9.0 x 106
4.1 x 103
2
15.9
6.4 x 106
3.46
3
15.0
7.9 x 106
Avg. (SD)
15.7 (0.6)
7.76 (1.3) x 106
1
12.8
4.9 x 105
4.1 x 102
2
13.1
4.6 x 105
3.06
3
14.2
4.8 x 105
Avg. (SD)
13.4 (0.7)
4.77 (0.14) x 10s
1
16.4
6.0 x 104
4.1x 101
2
16.7
5.5 x 104
3.43
3
16.2
2.9 x 104
Avg. (SD)
16.4 (0.2)
4.79 (1.6) x 104
1
12.7
8.2 x 103
4.1
2
12.8
4.7 x 103
3.51
3
13.0
4.3 x 103
26
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Starting Cell
Density
(CFU/mL)
Sample
Replicate*
ACt (T^T36)
T36 Cell Density
Avg. CFU/mL
Cell Density
Log Change
Avg. (SD)
12.8 (0.2)
5.73 (2.2) x 103
* Average (Avg.) and standard deviation (SD) for triplicate samples. Data are from manual DNA extraction.
Acronyms: CFU, colony-forming units; Or. cycle threshold.
incubation. For 30- (Table 11) or 36-hour (Table 12) incubation, the average ACt did not appear
to be a function of the starting cell density. The To Ct values ranged from -34-36.3 for the 103
CFU/mL level and -36.7-39 for the 102 CFU/mL level, with non-detect PCR results for two
lower starting cell densities (data not shown).
A replicate experiment was conducted following the same parameters including the same
incubation periods. In this case, the starting cell densities ranged from -5.7 to 5.7 x 103 per mL.
The culture and RV-PCR results are shown in Table 13 for the 30-h incubation and Table 14 for
the 36-h incubation. From culture analysis, the average log differences from To to T30 and from
To to T36 ranged from -2.50 to 2.79 and 2.86 to 3.25, respectively. The log change values were
slightly lower than the first replicate experiment for the 36-h incubation, although values were
higher than the 30-h incubation for this experiment, as expected. Results showed ACt values
ranging from 10.1 to 13.3 for the 30-h incubation and from 9.9 to 15.9 for the 36-h incubation.
As in the previous experiment, the average ACt did not vary as a function of the starting cell
density. In this case, the To Ct values ranged from -31.5-33 for the 103 CFU/mL level, -36-37
for the 102 CFU/mL level, 38-40 for the 101 CFU/mL level, and non-detect PCR results for the
10° CFU/mL level (data not shown). Together these data suggested that the 30-h incubation
period would be suitable for testing potential PCR and growth inhibitors as part of Task 3, since
differences in ACt values compared with the control treatment could more easily be observed if
there was an effect.
Table 13. RV-PCR and Culture Results for F. tularensis Schu S4 Cells Incubated for 30
Hours - Replicate Experiment
Starting Cell
Density (CFU/mL)
Sample
Replicate*
ACt (To — T30)
T30 Cell Density
Avg. CFU/mL
Cell Density Log
Change
1
11.8
3.7 x 106
5.7 x 103
2
12.2
2.5 x 106
2.70
3
12.2
2.4 x 106
Avg. (SD)
12.0 (0.2)
2.85 (0.73) x 106
1
11.0
1.9 x 105
5.7 x 102
2
11.5
2.0 x 105
2.5
3
10.1
1.6 x 105
Avg. (SD)
10.9 (0.7)
1.83 (0.22) x 10s
1
10.2
2.2 x 104
5.7x 101
2
11.5
2.5 x 104
2.61
3
10.3
2.3 x 104
Avg. (SD)
10.7 (0.7)
2.35 (0.12) x 104
5.7
1
13.3
4.3 x 103
2.79
27
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Starting Cell
Density (CFU/mL)
Sample
Replicate*
ACt (To — T30)
T30 Cell Density
Avg. CFU/mL
Cell Density Log
Change
2
12.5
3.5 x 103
3
12.4
2.8 x 103
Avg. (SD)
12.7 (0.5)
3.56 (0.75) x 103
* Average (Avg.) and standard deviation (SD) for triplicate samples.
Acronyms: CFU, colony-forming units; Or. cycle threshold.
Table 14. RV-PCR and Culture Results for F. tularensis Schu S4 Cells Incubated for 36
Hours - Replicate Experiment
Starting Cell
Density (CFU/mL)
Sample
Replicate*
ACx (T^T36)
T36 Cell Density
Avg. CFU/mL
Cell Density Log
Change
1
13.5
1.2 x 107
5.7 x 103
2
12.6
6.6 x 106
3.21
3
13.0
9.3 x 106
Avg. (SD)
13.0 (0.5)
9.20 (2.5) x 106
1
13.0
1.0 x 106
5.7 x 102
2
12.8
7.9 x 105
3.25
3
13.6
1.2 x 106
Avg. (SD)
13.1 (0.4)
1.02 (0.22) x 106
1
9.9
4.6 x 104
5.7x 101
2
10.3
3.3 x 104
2.86
3
11.3
4.5 x 104
Avg. (SD)
10.5 (0.7)
4.12 (0.71) x 104
1
14.5
4.7 x 103
5.7
2
14.8
5.8 x 103
2.89
3
15.9
2.8 x 103
Avg. (SD)
15.1 (0.7)
4.43 (1.5) x 103
* Average (Avg.) and standard deviation (SD) for triplicate samples.
Acronyms: CFU, colony-forming units; Or. cycle threshold.
4.2 Task 2. Evaluate manual and automated DNA extraction-purification procedures for
F. tularensis
4.2.1 Objectives
The objective for this task was to compare manual and automated DNA extraction protocols with
the goal of using the latter for more rapid analysis. For this task, a second aliquot was taken
during sample processing after 30- or 36-h incubation and frozen for subsequent automated DNA
extraction. PCR results were then to be compared for rapidity, precision, lower Ct, and labor to
the results from the first aliquot processed by manual extraction (reported for Task 1). These
aliquots contained 103 to 104 cells per mL determined from culture analysis. To extract and
analyze DNA from these aliquots containing virulent F. tularensis Schu S4 cells, equipment
including a Roche MagNA Pure® Compact and ABI 750 Fast PCR instrument were moved into
28
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the BSL3 laboratory. Since the Roche instrument was too large to be contained in a BSC, an
inactivation protocol needed to be validated to move crude cell lysates outside the BSC to
complete the DNA extraction process. According to LLNL biosafety officials, this protocol
constituted a removal from containment (per the CDC) although the robot was in the same
laboratory. The inactivation protocol was validated and documented prior to processing samples
from experiments (see section 2.8).
4.2.2 Procedure for F. tularensis Schu S4 Inactivation for Automated DNA Extraction in the
BSL3 Laboratory
For validation of the inactivation protocol prior to putting the samples on the automated DNA
extraction platform (outside the BSC), triplicates of both inactivated samples and positive
controls (not inactivated) were tested in three separate experiments. In these tests, 100% of the
inactivated material (or controls) were plated, with plates incubated under optimal growth
conditions for 96 h. An additional experiment was required by the CDC to assess whether the
chemicals used to inactivate cells were sufficiently washed from the samples (by repeated
centrifugation and resuspension) prior to culture analysis. In this case, the inactivated samples
were split in half after wash steps, with half plated directly and the other half spiked with a
known amount of F. tularensis cells prior to plating (where positive growth would indicate
sufficient removal of the inactivating chemicals). The three experiments used aliquots containing
~ 3-8 x 108 CFU/mL, which was concentrated to 200 |iL for inactivation.
After reviewing preliminary data on cell inactivation with the initial chemical lysis (30 min at
room temperature with mixing every 5 min), a step was added to perform heat lysis at 70°C for
10 min prior to conducting the Roche chemical lysis protocol. Using the modified inactivation
protocol and plating the entire treated and washed suspension showed no growth for triplicate
inactivated samples for triplicate experiments. Positive controls showed expected results
throughout. The testing showed that wash steps effectively removed the chemicals for
inactivation since added F. tularensis cells showed growth, whereas directly plated inactivated
(washed) cells had no growth.
4.2.3 Evaluation of Manual and Automated DNA Extraction-Purification Procedures Using
10s and 104 F. tularensis Schu S4 CFU/mL
For this task, aliquots taken from the RV-PCR experiments after either 30 or 36 h were extracted
by two different DNA extraction methods followed by real-time PCR analysis. At the incubation
endpoint, two 500 |iL aliquots were taken, centrifuged to pellet the cells, and then 300 |iL of the
supernatant was removed prior to storing at -20°C. Specifically, aliquots that had between 103
and 104 cells based on culture plating analysis were selected to compare the manual Promega and
automated Roche protocols. In this way, both methods could be evaluated using replicate
aliquots with the same cell densities. The manual Promega protocol was used as previously
described (US EPA, 2016), with one fewer lysis step and one fewer ethanol wash step compared
to the B. anthracis protocol. For the Roche DNA extraction protocol, the same method used by
the LRN on the MagNA Pure® Compact system was used. However, a 10-min heat lysis step
(70°C) preceded the addition of bacterial lysis buffer and incubation for 30 min. The data for
PCR analysis using the F5 F. tularensis-specific assay are shown in Table 15 and Table 17 for
29
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30-h incubation and Table 16 and Table 18 for 36-h incubation. The tables list the initial and
final cell densities in CFU/mL and PCR data in triplicate for both Promega and Roche DNA
extracts, with averages and standard deviations.
The data showed that the Roche DNA extraction protocol recovered more amplifiable DNA than
the Promega DNA extraction protocol, with lower Ct values evident for Roche DNA extracts.
Therefore, the Roche DNA extraction method was more sensitive than the Promega DNA
extraction method. For the first experiment (Table 15), the average difference between Ct
values ranged from 2.9 ± 0.2 to 3.2 ±1.0 for starting cell densities of 41 and 4.1 CFU/mL,
respectively (for 30-h incubation). For 36-h incubation (Table 16), the average Ct differences
ranged from 1.9 ± 0.3 to 1.7 ± 0.5 for starting cell densities of 41 and 4.1 CFU/mL, respectively.
For the second experiment (Table 17), the average Ct differences were lower, ranging from 1.0
± 0.3 to 1.6 ± 0.2 for 57 and 5.7 CFU/mL starting cell densities, respectively (for 30-h
incubation). For 36-h incubation (Table 18), average Ct differences ranged from 1.2 ± 0.3 to 0.8
±0.8 for these starting cell densities. The large standard deviation of 0.8 was due to one replicate
for the 5.7 CFU/mL sample set showing no difference in average Ct difference between
methods. Overall, the Roche DNA extracts showed an average 1.8 ± 0.9 lower Ct values than the
Promega DNA extracts over the four experiments shown in Tables 15-18. Only undiluted
extracts were analyzed since the extracts were from clean samples (only cells and media
components), and no PCR inhibition was expected. The data from undiluted extracts was deemed
sufficient to compare the two extraction methods, without requiring additional analysis.
In addition to better performance, the Roche method is semi-automated and thus, was less labor-
intensive, requiring initial manual processing for heat lysis and off-robot lysis (pipetting and
incubation), followed by loading on the robot in batches of eight for automated processing.
Centrifugation of the sample would be required if numerous particulates were present to avoid
clogging or improper pipetting by the robot, potentially adding another step after heat lysis and
initial chemical lysis. In this study, no centrifugation was needed since the highest particulate
density was relatively low at 4 mg ATD/mL (Task 3).
30
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Table 15. Comparison of Promega and Roche DNA Extraction-Purification Procedures for
Detection of F. tularensis Schu S4 Cells by PCR After 30-Hour Incubation
Avg. Initial
Avg. T3o
CFU/mL
CFU/mL
Sample
PCR
Promega
Roche
Ct T30
(from
(from
Replicate
Replicate
Ct T30
Ct T30
Difference*
inoculum)
plating)
1
31.5
28.9
1.3 (0.4)
1
2
31.7
28.7
2.9
x 104
3
31.7
28.8
Avg. (SD)
31.7(0.1)
28.8 (0.1)
1
32.4
29.2
7.8 (1.3)
2
2
33.0
29.6
3.1
41
x 103
3
32.3
29.6
Avg. (SD)
32.6 (0.4)
29.5 (0.2)
1
32.6
30.0
8.3 (0.4)
3
2
32.7
29.8
2.8
x 103
3
32.7
29.8
Avg. (SD)
32.7 (0.08)
29.9 (0.1)
9.5 (3.0)
x 103
Overall Avg. (SD)
32.3 (0.5)
29.4 (0.5)
2.9 (0.2)
1
36.2
32.8
1.2 (0.3)
1
2
35.2
32.5
2.9
x 103
3
35.2
32.6
Avg. (SD)
35.5 (0.6)
32.6 (0.2)
1
36.2
32.8
4.1
9.0 (3.0)
2
2
37.0
32.0
4.2
x 102
3
37.0
32.6
Avg. (SD)
36.7 (0.4)
32.5 (0.4)
1
35.0
33.2
1.4 (0.1)
3
2
36.0
32.5
2.3
x 103
3
34.6
32.9
Avg. (SD)
35.2 (0.7)
32.9 (0.3)
1.2 (0.3)
x 103
Overall Avg. (SD)
35.8 (0.9)
32.6 (0.3)
3.2 (1.0)
*Ct value from Promega DNA extraction minus the Ct value from Roche DNA extraction.
Acronyms: Avg. Average; SD, standard deviation; CFU, colony-forming units; Ct, cycle threshold.
31
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Table 16. Comparison of Promega and Roche DNA Extraction-Purification Procedures for
Detection of F. tularensis Schu S4 Cells by PCR After 36-Hour Incubation
Avg. Initial
CFU/mL
(from
inoculum)
Avg. T36
CFU/mL
(from
plating)
Sample
Replicate
PCR
Replicate
Promega
Ct T36
Roche
Ct T36
Ct T30
Difference*
1
28.7
26.8
6.0 (0.9)
1
2
28.6
26.8
1.7
x 104
3
28.6
27.0
Avg. (SD)
28.6 (0.06)
26.9 (0.1)
1
28.2
26.4
5.5 (0.8)
2
2
28.4
26.5
1.8
41
x 104
3
28.4
26.6
Avg. (SD)
28.3 (0.1)
26.5 (0.09)
1
28.8
26.5
2.9 (1.4)
3
2
28.8
26.6
2.2
x 104
3
28.7
26.7
Avg. (SD)
28.8 (0.08)
26.6 (0.07)
4.8 (1.7)
x 104
Overall Avg. (SD)
28.6 (0.2)
26.7 (0.2)
1.9 (0.3)
1
32.5
31.1
8.2 (0.1)
1
2
32.1
31.0
1.2
x 103
3
32.5
31.1
Avg. (SD)
32.3 (0.2)
31.1 (0.08)
1
32.3
30.2
4.1
4.7 (0.7)
2
2
32.0
30.1
2.1
x 103
3
32.2
30.1
Avg. (SD)
32.2 (0.1)
30.1 (0.06)
1
31.7
30.1
4.3 (0.8)
3
2
32.1
30.5
1.6
x 103
3
32.3
30.7
Avg. (SD)
32.0 (0.3)
30.4 (0.3)
5.7 (1.9)
x 103
Overall Avg. (SD)
32.2 (0.3)
30.5 (0.5)
1.7 (0.5)
*Ct value from Promega DNA extraction minus the Ct value from Roche DNA extraction.
Acronyms: Avg. Average; SD, standard deviation; CFU, colony-forming units; Ct, cycle threshold.
32
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Table 17. Comparison of Promega and Roche DNA Extraction-Purification Procedures for
Detection of F. tularensis Schu S4 Cells by PCR After 30-Hour Incubation - Replicate
Experiment
Avg. Initial
Avg. T3o
CFU/mL
CFU/mL
Sample
PCR
Promega
Roche
Ct T30
(from
(from
Replicate
Replicate
Ct T30
Ct T30
Difference*
inoculum)
plating)
1
28.9
28.2
2.2 (0.2) x
1
2
28.9
28.3
0.7
104
3
28.9
28.1
Avg. (SD)
28.9 (0.04)
28.2 (0.1)
1
28.9
27.9
2.5 (0.4) x
2
2
28.8
27.9
1.0
57
104
3
28.8
27.8
Avg. (SD)
28.8 (0.05)
27.9 (0.04)
1
28.6
27.3
2.3 (0.3) x
3
2
28.7
27.3
1.3
104
3
28.6
27.3
Avg. (SD)
28.6 (0.05)
27.3 (0.01)
2.4 (0.3)
x 104
Overall Avg. (SD)
28.8 (0.1)
27.8 (0.4)
1.0 (0.3)
1
31.5
30.5
4.3 (0.6) x
1
2
31.9
„**
1.3
103
3
31.6
30.2
Avg. (SD)
31.7 (0.2)
30.4 (0.2)
1
32.5
30.7
3.5 (0.4) x
2
2
32.5
30.9
1.7
5.7
103
3
32.6
30.6
Avg. (SD)
32.5 (0.1)
30.8 (0.1)
1
32.7
30.8
2.8 (0.2) x
3
2
32.7
30.9
1.7
103
3
32.5
30.9
Avg. (SD)
32.6 (0.1)
30.9 (0.05)
3.6 (0.7)
x 103
Overall Avg. (SD)
32.3 (0.5)
30.7 (0.3)
1.6 (0.2)
*Ct value from Promega DNA extraction minus the Ct value from Roche DNA extraction.
**Only two of three PCR replicates provided valid data.
Acronyms: Avg. Average; SD, standard deviation; CFU, colony-forming units; Ct, cycle threshold.
33
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Table 18. Comparison of Promega and Roche DNA Extraction-Purification Procedures for
Detection of F. tularensis Schu S4 Cells by PCR After 36-Hour Incubation - Replicate
Experiment
Avg. Initial
Avg. T36
CFU/mL
CFU/mL
Sample
PCR
Promega
Roche
Ct T36
(from
(from
Replicate
Replicate
Ct T36
Ct T36
Difference*
inoculum)
plating)
1
28.2
27.2
2.4 (0.5) x
1
2
28.1
27.0
1.0
104
3
28.2
27.2
Avg. (SD)
28.1
27.1
1
28.0
27.0
2.5 (0.8) x
2
2
28.2
27.0
1.1
57
104
3
28.1
26.9
Avg. (SD)
28.1
27.0
1
27.7
26.3
3.2 (1.4) x
3
2
27.8
26.3
1.5
104
3
27.7
26.4
Avg. (SD)
27.8
26.3
2.7 (0.9)
x 104
Overall Avg. (SD)
28.0 (0.2)
26.8 (0.4)
1.2 (0.3)
1
30.5
29.6
4.7 (3.0) x
1
2
30.4
29.5
1.0
103
3
30.5
29.6
Avg. (SD)
30.5
29.5
1
30.2
28.6
5.4 (1.5) x
2
2
30.1
28.8
1.5
5.7
103
3
30.2
28.6
Avg. (SD)
30.2
28.7
1
29.0
29.0
5.5 (1.6) x
3
2
29.3
29.1
0.0
103
3
29.1
29.2
Avg. (SD)
29.1
29.1
5.2 (1.9)
x 103
Overall Avg. (SD)
30.0 (0.6)
29.1 (0.4)
0.8 (0.8)
*Ct value from Promega DNA extraction minus the Ct value from Roche DNA extraction.
Acronyms: Avg. Average; SD, standard deviation; CFU, colony-forming units; Ct, cycle threshold.
4.3 Task 3. Evaluate and optimize an RV-PCR protocol for F. tularensis Schu S4 detection
in water samples with relevant challenges
4.3.1 Objectives
In this task, RV-PCR method performance with regard to method inhibition, analytical
sensitivity of detection, and time-to-results, was evaluated with a limited range of conditions
under which samples are likely to be obtained, including samples with both soluble and insoluble
contaminants. Iron sulfate and humic acid levels were selected that were representative (although
34
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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 are included in Materials and Methods Section 2.4.
4.3.2 Evaluation of chemical and biological challenges for RV-PCR analysis of F. tularensis
Schu S4 in water samples
In previous experiments, PBS buffer was used as a surrogate for water. Challenges included
debris containing non-target microorganisms represented by native (non-autoclaved) ATD, as
characterized by Rose et al. (2011) used at 4 mg/mL or 12 mg/3 mL sample (with ~5 x 103
CFU/mg ATD according to Rose et al.). Control treatments used PBS buffer without debris. In
addition, ferrous sulfate (10 |Lxg Fe2+/mL) and humic acid (50 |j,g/mL) were tested together since
iron minerals and organic matter such as humic acid are often present in environmental samples
including water samples, and the iron minerals and organic matter are known PCR inhibitors
(Schrader et al., 2012; Sidstedt et al., 2015). The /•'. tularensis Schu S4 cell suspensions were
prepared as described for previous experiments (for Tasks 1 and 2) and held at 4°C for 24 hours
to mimic sampling and storage at 4°C during shipment prior to analysis. The 30-h incubation
period was used based on the previous experiments to obtain measurable Ct values even if
growth and/or PCR inhibition were observed in the presence of the soluble or insoluble challenge
materials.
The results for three different starting F. tularensis Schu S4 cell levels (CFU/mL) for the first
experiment are shown in Table 19, Table 20, and Table 21 for average cell densities of-1,200,
120, and 12 CFU/3-mL sample, respectively. The results are shown as To Ct, T30 Ct, and ACt.
As mentioned in Sections 2.11 and 2.12, if two or three of three PCR replicates were NDT, then
a Ct value of 45 (i.e., the number of PCR cycles run) was assigned as the average To Ct or T30
Ct (as appropriate) to calculate ACt; whereas, if two or three of three replicates showed positive
Ct values, the average of two or three replicate Ct values was used to calculate ACt. Results for
a replicate experiment with slightly higher cell densities of-2,500, 250, and 25 CFU/mL are
shown in Table 22, Table 23, and Table 24, respectively. Individual PCR values are shown as
well as the average and standard deviation for the replicate PCR analyses. In addition, 10-fold
dilutions of DNA extracts from the ATD treatments were included to check for PCR inhibition
(referred to as ATD 1:10 in the treatment column.
The T30 Ct data showed that PBS buffer with Fe and humics had no apparent inhibition with
lower T30 Ct values compared to clean controls (-2-7 Ct decrease depending on starting cell
number) for both replicate experiments. For the first experiment with the 103-cell level (-1,200
CFU/sample), the Fe/humics treatment showed ACt values that were more than double the
35
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Table 19. RV-PCR Results for F. tularensis Schu S4 Cells (-1,200 CFU/3-mL Sample) in
the Presence of Fe/Humics or ATD
Treatment
Sample
Replicate
PCR
Replicate
To CT
T30 Ct
ACt (To—T30)
1
34.5
25.1
1
2
33.9
25.1
9.3
3
34.4
25.0
Avg. (SD)
34.3 (0.3)
25.0 (0.04)
1
33.7
26.6
Control
2
2
33.8
26.6
7.4
3
34.5
26.6
Avg. (SD)
34.0 (0.5)
26.6 (0.03)
1
34.1
25.7
3
2
33.6
25.7
8.3
3
34.2
25.7
Avg. (SD)
34.0 (0.3)
25.7 (0.02)
1
NDT
22.3
1
2
NDT
22.3
22.7
3
NDT
22.3
Avg. (SD)
NDT
22.3 (0.04)
1
42.1
22.7
Fe/Humics
2
2
42.0
22.7
18.7
3
40.0
22.7
Avg. (SD)
41.4 (1.2)
22.7 (0.02)
1
37.3
21.6
3
2
35.7
21.6
14.5
3
35.4
21.6
Avg. (SD)
36.1 (1.0)
21.6 (0.01)
1
NDT
33.2
1
2
39.7
33.6
5.5
3
38.3
33.8
Avg. (SD)
39.0 (1.0)
33.5 (0.3)
1
38.2
33.1
ATD
2
2
38.8
NDT
5.3
3
NDT
33.4
Avg. (SD)
38.5 (0.4)
33.2 (0.2)
1
37.0
33.5
3
2
37.5
34.1
3.7
3
NDT
33.1
Avg. (SD)
37.2 (0.4)
33.5 (0.5)
1
38.7
36.6
1
2
NDT
37.4
2.1
3
39.0
36.0
Avg. (SD)
38.8 (0.3)
36.7 (0.7)
1
NDT
37.3
ATD
2
2
NDT
36.6
8.1
1:10*
3
NDT
36.6
Avg. (SD)
NDT
36.9 (0.4)
1
NDT
36.7
3
2
NDT
36.9
8.3
3
NDT
36.6
Avg. (SD)
NDT
36.7 (0.2)
* The same ATD concentration was used with the DNA extract analyzed after 10-fold dilution with PCR H20.
Acronyms: Avg. Average; SD, standard deviation; CFU, colony-forming units; Ci. cycle threshold.
-------
Table 20. RV-PCR Results for F. tularensis Schu S4 Cells (-120 CFU/3-mL Sample) in the
Presence of Fe/Humics or ATD
Treatment
Sample
Replicate
PCR
Replicate
To CT
T30 Ct
ACt (To—T30)
1
38.9
28.6
1
2
37.0
28.6
9.3
3
37.7
28.5
Avg. (SD)
37.9 (1.0)
28.6 (0.02)
1
37.0
28.2
Control
2
2
NDT
28.3
8.9
3
37.1
28.2
Avg. (SD)
37.1 (0.07)
28.2 (0.04)
1
36.7
28.2
3
2
36.6
28.0
9.0
3
37.7
28.3
Avg. (SD)
37.0 (0.6)
28.2 (0.1)
1
NDT
26.8
1
2
42.9
26.4
16.2
3
42.5
26.4
Avg. (SD)
42.7 (0.3)
26.5 (0.2)
1
NDT
27.0
Fe/Humics
2
2
NDT
26.8
18.3
3
NDT
26.2
Avg. (SD)
NDT
26.7 (0.4)
1
38.3
23.6
3
2
40.3
23.7
15.4
3
38.4
23.7
Avg. (SD)
39.0 (1.1)
23.7 (0.06)
1
NDT
38.7
1
2
NDT
36.2
7.2
3
NDT
38.5
Avg. (SD)
NDT
37.8 (1.4)
1
NDT
37.8
ATD
2
2
NDT
35.7
8.2
3
NDT
NDT
Avg. (SD)
NDT
36.8 (1.5)
1
NDT
36.7
3
2
NDT
37.0
8.2
3
NDT
36.8
Avg. (SD)
NDT
36.8 (0.1)
1
NDT
39.5
1
2
NDT
39.8
5.3
3
NDT
NDT
Avg. (SD)
NDT
39.7 (0.2)
1
NDT
NDT
ATD
2
2
NDT
NDT
0.0
1:10*
3
NDT
NDT
Avg. (SD)
NDT
NDT
1
NDT
NDT
3
2
NDT
NDT
0.0
3
NDT
NDT
Avg. (SD)
NDT
NDT
* The same ATD concentration was used with the DNA extract analyzed after 10-fold dilution with PCR H20.
Acronyms: Avg. Average; SD, standard deviation; CFU, colony-forming units; Ct. cycle threshold.
37
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Table 21. RV-PCR Results for F. tularensis Schu S4 Cells (~12 CFU/3-mL Sample) in the
Presence of Fe/Humics or ATD
Treatment
Sample
Replicate
PCR
Replicate
To CT
T30 Ct
ACt (To—T30)
1
NDT
35.3
1
2
NDT
33.9
10.3
3
NDT
34.8
Avg. (SD)
NDT
34.7 (0.7)
1
NDT
34.5
Control
2
2
NDT
34.3
10.9
3
NDT
33.5
Avg. (SD)
NDT
34.1 (0.6)
1
NDT
32.4
3
2
NDT
32.3
12.7
3
NDT
32.4
Avg. (SD)
NDT
32.3 (0.07)
1
NDT
27.3
1
2
NDT
27.2
17.8
3
NDT
27.1
Avg. (SD)
NDT
27.2 (0.07)
1
NDT
29.1
Fe/Humics
2
2
NDT
29.2
15.9
3
NDT
29.0
Avg. (SD)
NDT
29.1 (0.08)
1
NDT
28.5
3
2
NDT
28.5
16.5
3
NDT
28.6
Avg. (SD)
NDT
28.5 (0.07)
1
NDT
NDT
1
2
NDT
NDT
0.0
3
NDT
NDT
Avg. (SD)
NDT
NDT
1
NDT
NDT
ATD
2
2
NDT
NDT
0.0
3
NDT
NDT
Avg. (SD)
NDT
NDT
1
NDT
NDT
3
2
NDT
NDT
0.0
3
NDT
NDT
Avg. (SD)
NDT
NDT
1
NDT
NDT
1
2
NDT
NDT
0.0
3
NDT
NDT
Avg. (SD)
NDT
NDT
1
NDT
NDT
ATD
2
2
NDT
NDT
0.0
1:10*
3
NDT
NDT
Avg. (SD)
NDT
NDT
1
NDT
NDT
3
2
NDT
NDT
0.0
3
NDT
NDT
Avg. (SD)
NDT
NDT
* The same ATD concentration was used with the DNA extract analyzed after 10-fold dilution with PCR H20.
Acronyms: Avg. Average; SD, standard deviation; CFU, colony-forming units; Ci. cycle threshold.
-------
values for the clean controls since the To Ct values were higher than the values for the clean
controls. This treatment also showed more variability in average To Ct values between sample
replicates potentially due to PCR inhibition and slight differences in sample replicate
composition (e.g., pipetting variability), whereas T30 Ct values were similar between sample
replicates (21.6 - 22.7). For the other cell levels in the first experiment and for all cell levels in
the second experiment, the To Ct values were comparable between Fe/Humics and clean control
treatments, and higher ACt values were attributed to lower T30 Ct values.
It is possible that cells were limited for Fe or nutrients present with the humic acid formulation,
suggesting that improvements to the growth medium could enhance F. tularensis growth.
However, in the presence of competing microbes, the addition of Fe and humics may not
specifically enhance F. tularensis growth, and other microorganisms could out-compete F.
tularensis given its slower growth character. Growth inhibition was observed in the presence of
test dust (including background microbes) resulting in no detection at the 101 CFU/mL level
(-12-25 cells) for both replicate experiments (Table 21 and Table 24).
In general, PCR inhibition did not appear to contribute to higher Ct values since the difference
between T30 Ct values for undiluted and 1:10 diluted ATD DNA extracts showed roughly a three
Ct difference, as expected. In some cases (102-cell level for both experiments), the 10-fold
diluted DNA extracts showed non-detect To Ct results while values were measured for undiluted
extracts; these results are expected for data near the PCR assay detection limit. The 10-fold
diluted extracts showed higher ACt values in the 103-cell level for both experiments (Table 19
and Table 22), since the To Ct values were NDT and set to 45 (the number of PCR cycles run) to
calculate ACt. These data also suggest that a 30-h to 36-h incubation period should be used
rather than 24-h to improve detection of viable F. tularensis cells in complex samples containing
background microbes. The overall results for effect of the Fe/Humics and ATD on the RV-PCR
method are summarized in Table 25.
39
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Table 22. RV-PCR Results for F. tularensis Schu S4 Cells (-2,500 CFU/3-mL Sample) in
the Presence of Fe/Humics or ATD - Replicate Experiment
Treatment
Sample
Replicate
PCR
Replicate
To CT
T30 Ct
ACt (To—T30)
1
34.7
25.3
1
2
34.0
25.1
9.3
3
34.7
25.2
Avg. (SD)
34.4 (0.4)
25.2 (0.07)
1
34.2
26.2
Control
2
2
33.8
26.3
7.9
3
34.3
26.2
Avg. (SD)
34.1 (0.3)
26.2 (0.04)
1
33.6
26.2
3
2
33.4
26.1
7.2
3
33.0
26.1
Avg. (SD)
33.4 (0.3)
26.1 (0.04)
1
33.7
22.0
1
2
33.4
21.9
11.5
3
33.1
21.9
Avg. (SD)
33.4 (0.3)
21.9 (0.03)
1
33.8
20.5
Fe/Humics
2
2
34.0
20.6
13.3
3
33.7
20.6
Avg. (SD)
33.8 (0.1)
20.5 (0.02)
1
37.5
19.9
3
2
37.8
19.9
17.2
3
36.3
20.0
Avg. (SD)
37.2 (0.8)
20.0 (0.03)
1
37.6
38.3
1
2
39.6
39.3
-0.1
3
NDT
38.4
Avg. (SD)
38.6 (1.4)
38.7 (0.5)
1
38.0
37.5
ATD
2
2
38.8
35.1
2.4
3
37.8
34.8
Avg. (SD)
38.2 (0.6)
35.8 (1.5)
1
NDT
34.6
3
2
NDT
33.5
10.7
3
NDT
34.7
Avg. (SD)
NDT
34.3 (0.7)
1
NDT
35.8
1
2
NDT
36.3
9.2
3
NDT
35.1
Avg. (SD)
NDT
35.8 (0.6)
1
NDT
35.9
ATD
2
2
NDT
35.5
9.2
1:10*
3
NDT
36.1
Avg. (SD)
NDT
35.8 (0.3)
1
NDT
37.8
3
2
NDT
36.1
8.3
3
NDT
36.1
Avg. (SD)
NDT
36.7 (1.0)
* The same ATD concentration was used with the DNA extract analyzed after 10-fold dilution with PCR H20.
Acronyms: Avg. Average; SD, standard deviation; CFU, colony-forming units; Ci. cycle threshold.
-------
Table 23. RV-PCR Results for F. tularensis Schu S4 Cells (-250 CFU/3-mL Sample) in the
Presence of Fe/Humics or ATD - Replicate Experiment
Treatment
Sample
Replicate
PCR
Replicate
To CT
T30 Ct
ACt (To—T30)
1
NDT
29.9
1
2
38.3
29.7
7.8
3
36.8
29.8
Avg. (SD)
37.6 (1.1)
29.8 (0.1)
1
38.8
28.9
Control
2
2
36.3
29.2
8.1
3
36.3
28.9
Avg. (SD)
37.1 (1.4)
29.0 (0.1)
1
36.1
29.6
3
2
37.0
29.5
6.8
3
35.7
29.2
Avg. (SD)
36.3 (0.7)
29.4 (0.2)
1
37.2
24.9
1
2
37.8
24.9
12.5
3
37.2
24.8
Avg. (SD)
37.4 (0.3)
24.9 (0.05)
1
38.7
25.5
Fe/Humics
2
2
38.4
25.5
12.6
3
37.1
25.5
Avg. (SD)
38.1 (0.9)
25.5 (0.01)
1
38.0
24.9
3
2
NDT
24.8
13.9
3
39.4
24.8
Avg. (SD)
38.7 (1.0)
24.8 (0.08)
1
NDT
38.4
1
2
NDT
37.3
7.5
3
NDT
36.7
Avg. (SD)
NDT
37.5 (0.9)
1
NDT
40.6
ATD
2
2
NDT
40.3
5.2
3
NDT
38.5
Avg. (SD)
NDT
39.8 (1.1)
1
NDT
37.6
3
2
NDT
39.2
7.0
3
NDT
37.2
Avg. (SD)
NDT
38.0 (1.1)
1
NDT
NDT
1
2
NDT
NDT
0.0
3
NDT
NDT
Avg. (SD)
NDT
NDT
1
NDT
NDT
ATD
2
2
NDT
38.0
6.4
1:10*
3
NDT
39.2
Avg. (SD)
NDT
38.6 (0.8)
1
NDT
NDT
3
2
NDT
NDT
0.0
3
NDT
NDT
Avg. (SD)
NDT
NDT
* The same ATD concentration was used with the DNA extract analyzed after 10-fold dilution with PCR H20.
Acronyms: Avg. Average; SD, standard deviation; CFU, colony-forming units; Ci. cycle threshold.
41
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Table 24. RV-PCR Results for F. tularensis Schu S4 Cells (~25 CFU/3-mL Sample) in the
Presence of Fe/Humics or ATD - Replicate Experiment
Treatment
Sample
Replicate
PCR
Replicate
To CT
T30 Ct
ACt (To—T30)
1
NDT
34.3
1
2
NDT
34.7
10.6
3
NDT
34.0
Avg. (SD)
NDT
34.4 (0.4)
1
NDT
33.6
Control
2
2
NDT
33.6
11.4
3
NDT
33.6
Avg. (SD)
NDT
33.6 (0.02)
1
NDT
32.6
3
2
NDT
32.9
12.3
3
NDT
32.6
Avg. (SD)
NDT
32.7 (0.2)
1
NDT
28.5
1
2
NDT
28.6
16.4
3
NDT
28.6
Avg. (SD)
NDT
28.6 (0.08)
1
NDT
28.3
Fe/Humics
2
2
NDT
28.2
16.8
3
NDT
28.1
Avg. (SD)
NDT
28.2 (0.07)
1
NDT
28.6
3
2
NDT
28.4
16.5
3
NDT
28.4
Avg. (SD)
NDT
28.5 (0.08)
1
NDT
44.4
1
2
NDT
43.3
1.2
3
NDT
NDT
Avg. (SD)
NDT
43.8 (0.8)
1
NDT
NDT
ATD
2
2
NDT
NDT
0.0
3
NDT
NDT
Avg. (SD)
NDT
NDT
1
NDT
41.4
3
2
NDT
NDT
4.9
3
NDT
38.8
Avg. (SD)
NDT
40.1 (1.8)
1
NDT
NDT
1
2
NDT
NDT
0.0
3
NDT
NDT
Avg. (SD)
NDT
NDT
1
NDT
NDT
ATD
2
2
NDT
NDT
0.0
1:10*
3
NDT
NDT
Avg. (SD)
NDT
NDT
1
NDT
NDT
3
2
NDT
NDT
0.0
3
NDT
NDT
Avg. (SD)
NDT
NDT
* The same ATD concentration was used with the DNA extract analyzed after 10-fold dilution with PCR H20.
Acronyms: Avg. Average; SD, standard deviation; CFU, colony-forming units; Ci. cycle threshold.
-------
Table 25. Summary of RV-PCR Results Based on Condition and Starting Cell Level
(Tables 21-24)
Condition
Approximate
Starting CFU per
3-mL Sample
ACt Range
Positive
RV-PCR Results
No Challenge
2500
7.2-9.3
3 of 3
1200
7.4-9.3
3 of 3
250
6.8-8.1
3 of 3
120
8.9-9.3
3 of 3
25
10.6-12.3
3 of 3
12
10.3-12.7
3 of 3
Fe/Humics
2500
11.5-17.2
3 of 3
1200
14.5-22.7
3 of 3
250
12.5-13.9
3 of 3
120
15.4-18.3
3 of 3
25
16.4-16.8
3 of 3
12
15.9-17.8
3 of 3
ATD
(undiluted or
10-fold diluted)*
2500
9.2-10.7
3 of 3
1200
5.5-8.3
2 of 3
250
6.4-7.5
3 of 3
120
7.2-8.2
3 of 3
25
0-4.9
Oof 3
12
0-0
Oof 3
* The ACt range includes the highest ACt value per ATD sample replicate, either from undiluted or 10-fold diluted
DNA extracts.
Highlighted cells represent conditions and starting cell numbers where three of three sample replicates had positive
RV-PCR results.
Acronyms: ATD, Arizona Test Dust; Or. cycle threshold.
43
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5.0 Summary and Conclusions
In summary, an optimized RV-PCR method was developed for detection of viable F. tularensis
cells from water samples, which along with the Y. pestis RV-PCR method (US EPA, 2016),
could serve as a model method for the detection of vegetative bacterial pathogens. The RV-PCR
method was optimized by improving procedures for high-throughput culturing (in 48-well plates)
and DNA extraction/purification using an automated platform. The optimized method was
evaluated with regard to sensitivity and performance with complex water samples, including
both biological and chemical challenges.
Specifically, previous work on /•'. tularensis growth optimization (Morris et al., 2017) using a
BHI-Vitox-Fildes-Histidine (BVFH) medium was leveraged for development of 6X-concentrated
growth medium for RV-PCR analysis. The 6X BVFH medium diluted to IX supported F.
tularensis cell growth comparable to the growth observed for the IX BVFH medium; values
were within -72-75% of the cell growth that for IX BVFH medium (p = 0.01 - 0.18, two-tailed,
pairwise t-test). However, 10X BVFH diluted to IX did not support adequate growth due to
precipitation of medium components. Concentration of the medium enabled use of a larger
volume water sample (3-mL plus 0.6 mL 6X BVFH medium per 5-mL well) and demonstrated
reproducible growth even at low inoculum levels (<10 cells per mL).
Prior to RV-PCR method evaluation, real-time PCR assay optimization showed that addition of
1.25 U Platinum™ Taq Polymerase to the reaction resulted in better sensitivity with average Ct
values reduced with the enzyme addition by an average 9.8 and 8.9 Ct units for F4 and F5
assays, respectively. Therefore, the additional enzyme was included along with the AmpliTaq
Gold® DNA polymerase from the MasterMix.
Testing was also performed to determine whether storing the cells at 4°C for 24 h impacted RV-
PCR analysis (and the corresponding cell growth), thus mimicking conditions if samples were
shipped overnight at 4°C for processing the next day. Using a 24-h incubation period for RV-
PCR analysis of clean samples, two replicate experiments with starting cell levels ranging from
45 to 7,000 CFU/mL showed that holding cells for 24 h prior to analysis led to slightly lower
increases in log CFU/mL; however, results were significant only for a couple of cases (p <
0.01),) and corresponding ACt values were not significantly different between treatments.
Overall, the data suggested that the assay sensitivity for viable F. tularensis cells using 24-h
incubation for RV-PCR analysis was in the range of 135 to 210 cells per 3-mL for clean water
samples.
Challenge testing with potential soluble (iron, humic acid) and insoluble chemical interferences
(metal oxides in ATD) and live, non-target biological interferences (in ATD) addressed a range
of potential "real world" complex sample types. Based on the testing with ATD (12 mg/3-mL
sample), it is possible that extending the incubation time to 36 h might reduce the risk of false
negative results for samples with low target cell numbers (101 CFU level or 10-99 cells per 3-mL
sample) and high non-target microbial backgrounds (> 104—105 CFU/3-mL sample); however,
30-h incubation was sufficient for water samples with potential chemical inhibitors including
iron (up to 10 |ig/mL) and humic acid (up to 50 |ig/mL). For these samples, the T30 Ct values
were approximately 3.2 - 7.5 units lower than the clean controls (PBS). Growth of F. tularensis
44
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cells may have been enhanced by iron and/or nutrients associated with the humic acid
formulation. These data suggest that the growth medium could be further optimized, although
this could also enhance growth of competing microbes. Conversely, in the presence of ATD, F.
tularensis growth appeared to be inhibited for low cell levels (101 CFU/3-mL sample) leading to
non-detect results, which could not be attributed to PCR inhibition because both undiluted and
10-fold diluted DNA extracts were non-detect. Additional testing is needed to assess RV-PCR
method performance with real-world water samples as well as with a dead F. tularensis cell
background for natural decay or post-decontamination scenarios.
Together, these findings showed that the RV-PCR method could be applied to F. tularensis in
water samples containing potential inhibitors, demonstrating good sensitivity and method
performance with complex sample matrices. While traditional culture methods are considered the
gold standard for viability analysis, this effort demonstrated that RV-PCR could detect 101 to
102-cell level per 3-mL sample in < 36 h (sample processing and analysis time) compared to
more than 72 h required for confirmed results from plate culture analysis. In addition, the RV-
PCR method would produce significantly less waste and have a smaller laboratory space
footprint for analysis. To illustrate these characteristics, RV-PCR uses a single 48-well plate for
48 samples (and controls) for growth compared with the culture method that uses 11 Chocolate
Agar plates, dilution tubes, and an enrichment culture tube per sample, with additional plates to
re-streak from the enrichment culture for target colony isolation.
In addition, other manual or automated DNA extraction platforms could be integrated into the
RV-PCR method. An advantage with the Promega MagneSil® procedure used in this effort was
that it required less centrifugation compared to other manual protocols (i.e., Qiagen QIAamp®).
Centrifugation is more time-consuming under BSL-3 conditions, requiring transfer of the rotor to
the BSC for sample processing. In addition, the Roche automated protocol could enable use of
existing MagNA Pure® platforms in LRN laboratories, thus providing a more rapid analysis with
more reproducible RV-PCR results. In this study, DNA extracts from the Roche MagNA Pure®
Compact showed an average 1.8 ± 0.9 lower Ct values than the manual Promega MagneSil®
DNA extracts. The real-time PCR assays used in this effort consistently demonstrated < 30
genome equivalent sensitivity; however, other assays in use for F. tularensis detection could
readily be integrated into the RV-PCR method.
An SOP was developed (Appendix A) providing specific details for conducting sample
processing and analysis as part of a tularemia incident response. This SOP can interface with a
front-end sampling processing/concentration procedure such as ultrafiltration (e.g., Francy et al.,
2009) and secondary filtration, like the procedure used for Y. pestis (US EPA, 2016), although
this requires further testing for relevant filtrate samples. The RV-PCR method for detection of
viable F. tularensis from water samples will help enhance the WLA capability for rapid, reliable,
and high-throughput sample analysis to effectively respond to an intentional, accidental, or
natural outbreak incident resulting in water infrastructure contamination. Additionally,
subsequent to following sample-type-specific processing and concentration steps, this RV-PCR
method can be used not just for water samples, but also for other sample types during a response
to a wide-area tularemia incident.
45
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6.0 References
Abd, H., T. Johansson, I. Golovliov, G. Sandstrom, and M. Forsman. 2003. Survival and Growth
of Francisella tularensis in Acanthamoeba castellanii. Appl. Environ. Microbiol.
69(l):600-606.
Anda, P., J. Segura del Poz, J. Diaz, M. Diaz Garcia, R. Escudero, F. Garcia, J. Pena, M. Lopez,
C. Lopez Velasco, R. Sellek, M. Jimenez Chillaron, L. Sanchez Serrano, and J. Martinez
Navarro 2001. Waterborne Outbreak of Tularemia Associated with Crayfish Fishing.
Emerg. Infect. Dis. 7(No. 3 Supplement):575-582.
Berrada, Z.L. and S.R. Telford III. 2011. Survival of Francisella tularensis Type A in brackish
water. Arch. Microbiol. 193 (3): 223-226.
Dennis, D.T., T.V. Inglesby, D.A. Henderson, J.G. Bartlett, M.S. Ascher, E. Eitzen, A.D. Fine,
A.M. Friedlander, J. Hauer, M. Layton, S.R. Lillibridge, J.E. McDade, M.T. Osterholm,
T. O'Toole, G. Parker, T.M. Perl, P.K. Russell, and K. Tonat. 2001. Tularemia as a
Biological Weapon: Medical and Public Health Management. J. Am. Med. Assoc.
285(21):2763-2773.
Forsman, M., E.W. Henningson, E. Larsson, T. Johansson, and G. Sandstrom. 2000. Francisella
tularensis does not manifest virulence in viable but non-culturable state. FEMS
Microbiol. Ecol. 31(3):217-224.
Francy, D.S., R.N. Bushon, A.M.G. Brady, E.E. Bertke, C.M. Kephart, C.A. Likirdopulos, B.E.
Mailot, F.W. Schaefer III, and H.D.A. Lindquist. 2009. Comparison of Traditional and
Molecular Analytical Methods for Detecting Biological Agents in Raw and Drinking
Water Following Ultrafiltration. J. Appl. Microbiol. 107(5): 1479-1491.
Giircan, S. 2014. Epidemiology of tularemia. Balkan Med. J. 31:3-10.
Jones, R.M., M. Nicas, A. Hubbard, M.D. Sylvester, and A. Reingold. 2005. The Infectious Dose
of Francisella tularensis (Tularemia). Appl. Biosafety 10(4):227-23 9.
Kugeler, K.J., R. Pappert, Y. Zhou, and J.M. Petersen. 2006. Real-time PCR Assays for
Francisella tularensis Types A and B. Emerg. Infect. Dis. 12(11): 1799-1801.
Letant, S.E., G.A. Murphy, T.M. Alfaro, J.R. Avila, S.R. Kane, E. Raber, T.M. Bunt, and S. Shah.
2011. Rapid-Viability PCR method for detection of live, virulent Bacillus anthracis in
environmental samples. Appl. Environ. Microbiol. 77(18): 6570-6578.
Morris, B.J., H.Y. Buse, N.J. Adcock, and E.W. Rice. 2017. A Novel Broth Medium for
Enhanced Growth of Francisella tularensis. Lett. Appl. Microbiol. 64(6): 394-400.
NRC. 1979. Iron. National Research Council. University Park Press, Baltimore, MD.
Potter, B.B., and J.C. Wimsatt. 2005. Method 415.3 Measurement of Total Organic Carbon,
Dissolved Organic Carbon and Specific UV Absorbance at 254 nm in Source Water and
Drinking Water. U.S. Environmental Protection Agency, Cincinnati, OH.
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Rice, E.W. 2015. Occurrence and Control of Tularemia in Drinking Water. J. Am. Water Works
Assoc. 107(10):E486 E486-E496.
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. Appl. Environ. Microbiol. 77(23):8355-8359.
Saslaw, S., H.T. Eigelsbach, J.A. Prior, H.E. Wilson, and S. Carhart. 1961. Tularemia Vaccine
Study, II: Respiratory challenge. Arch. Intern. Med. 107(5):702-714.
Schrader, C., A. Schielke, L. Ellerbroek, and R. Johne. 2012. PCR inhibitors - Occurrence,
Properties and Removal. J. Appl. Microbiol. 113(5): 1014-1026.
Sidstedt, M., L. Jansson, E. Nilsson, L. Noppa, M. Forsman, P. Radstrom, and J. Hedman. 2015.
Humic Substances Cause Fluorescence Inhibition in Real-Time Polymerase Chain
Reaction. Anal. Biochem. 487:30-37.
US EPA. 2015. Literature Review on Processing and Analytical Methods for Francisella
tularensis in Soil and Water. U.S. Environmental Protection Agency, Washington, DC.
US EPA. 2016. Protocol for Detection of Yersiniapestis in Environmental Samples During the
Remediation Phase of a Plague Incident. U.S. Environmental Protection Agency,
Cincinnati, OH.
US EPA, 2017. Sampling Guidance for Unknown Contaminants in Drinking Water. EPA-817-R-
08-003.
US EPA. 2017. Protocol for Detection of Bacillus anthracis in Environmental Samples During
the Remediation Phase of an Anthrax Incident. U.S. Environmental Protection Agency,
Cincinnati, OH.
Wai, S.N., Y. Mizunoe, A. Takade, and S.I. Yoshida. 2000. A comparison of solid and liquid
media for resuscitation of starvation- and low-temperature-induced nonculturable cells of
Aeromonas hydrophila. Arch. Microbiol. 173 301-310.
WHO. 2003. Iron in drinking-water. Background document for preparation of WHO Guidelines
for drinking-water quality. Geneva, World Health Organization
(WHO/SDE/WSH/03.04/08).
47
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Appendix A. Standard Operating Procedure -
Manual Protocol for Rapid Viability Polymerase Chain
Reaction (RV-PCR) for Analysis of Francisella tularensis in
Water Samples
48
-------
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 30 h
(to allow Francisella tularensis cell propagation prior to DNA extraction/purification and
analysis).
Acronyms
BHI Brain Heart Infusion
BSC biosafety cabinet
BSL Biosafety Level
B VFH BHI/Vitox/Fildes/Hi sti dine
BHI brain heart infusion
CA Chocolate Agar
CFU colony forming units
Ct cycle threshold
DD deionized, distilled
DNA deoxyribonucleic acid
EDTA ethylenediaminetetraacetic acid
hr hour
PAPR powered air purifying respirators
PBS phosphate buffered saline
PES polyethersulfone
PMPs paramagnetic particles
PPE personal protection equipment
RCF relative centrifugal force
RV-PCR rapid viability-polymerase chain reaction
TE Tris EDTA
To time 0, prior to incubation
T30 after 30 hours of incubation
TNTC too numerous to count
Trademarked Products
Tnuk'niiirk
Holder
l.ociilion
AeraSeal™
Excel Scientific, Inc.
Victorville, CA
Bacto™
BD Biosciences
Franklin Lakes, NJ
Biopur® Safe-Lock®
Eppendorf
Hamburg, Germany
Clorox®
Clorox Company
Oakland, CA
Costar®
Corning
Corning, NY
Dispatch®
Clorox Company
Oakland, CA
Dynamag™
Life Technologies
Carlsbad, CA
Lazy-L™
Millipore Sigma
Burlington, MA
49
-------
Tnuk'niiirk
Holder
l.ociilion
MagncSil
Promcga
Madison, \\ 1
MasterPure®
Epicentre Biotechnologies
Madison, WI
MagNA Pure®
Roche Diagnostics
Indianapolis, IN
MaxQ™
Thermo Scientific
Waltham, MA
Millipore®, Milli-Q™
Millipore Corp.
Billerica, MA
Oxoid®
Oxoid Limited
Cheshire, England
Remel™
Remel
Lenexa, KS
TaqMan®
Life Technologies
Carlsbad, CA
Tyvek® suit
DuPont
Wilmington, DE
Ziploc®
Johnson and Johnson
New Brunswick, NJ
50
-------
Laboratory set-up
• Don 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 [Ultra Clorox® Germicidal bleach;
6.15% sodium hypochlorite] + 9 volumes water). Date and label with initials.
• Clean/bleach biosafety cabinet (BSC) and bench surfaces allowing 30-minute (min)
contact time. Rinse with DD water or 70% isopropanol.
• 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)
• Ultra Clorox® Germicidal bleach (VWR Cat. No. 76245-190 or equivalent)
• Acetic Acid, Glacial (VWR Cat. No. CA71006-436 or equivalent)
• Ziploc®bags (large -20" (inches) 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, distilled (DD) 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
• Disposable Serological Pipettes 5 mL serological pipettes 5mL (VWR Cat. No.
89130-896 or equivalent)
• Disposable Serological Pipettes 10 mL serological pipettes lOmL (VWR Cat. No.
89130-898 or equivalent)
• Disposable Serological Pipettes 25 mL serological pipettes 25mL (VWR Cat. No.
89130-900 or equivalent)
51
-------
• Disposable Serological Pipettes 50 mL serological pipettes 50mL (VWR Cat. No.
89130-902 or equivalent)
• 500-mL bottles (Sigma-Aldrich Cat. No. 1395-500HTC or equivalent)
• 1-L bottles (Sigma-Aldrich Cat. No. 1395-1LHTC or equivalent)
Supplies for Rapid Viabilitv-Polymerase Chain Reaction (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. F187950001 or equivalent)
• Screw cap tubes, 2 mL (VWR Cat. No. 89004-298 or equivalent)
• 96-well microcentrifuge tube rack(s) for 2 mL tubes (8 x 12 layout) (Bel-Art, Cat.
No. F188450031 or equivalent)
• 2 mL Eppendorf tubes (Fisher Scientific Cat. No. 05-402-24C or equivalent)
• 48-well plates for rapid viability-polymerase chain reaction (RV-PCR) sample
incubation (E&K Scientific Cat. No. EK-2044 or equivalent)
• 0.2 micron Ultrafree-MC filter units (Millipore Cat. No. UFC30GV0S) for filtration
following DNA extraction
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, sterile (Teknova Cat. No. W3350 or equivalent)
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).
• Chocolate Agar plates (Hardy Diagnostics Cat. No. E14 or equivalent)
52
-------
Equipment
• Biosafety 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 or equivalent)
• 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 microcentrifuge for Eppendorf tubes with aerosol-tight rotor (Eppendorf,
Cat. No. 5415R or equivalent)
• 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 |j,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 35-37 °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)
• pH meter (VWR Cat. No. 89231-664 or equivalent)
Reagents
• PCR-grade water, sterile (Teknova Cat. No. W3350 or equivalent)
• MilliQ® H2O or equivalent
• Phosphate buffered saline (PBS) buffer (Teknova Cat. No. P0261 or equivalent)
• 10X PBS buffer (Teknova Cat. No. P0195 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)
53
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• Anti-Foam Reagent (VWR Cat. No. PAMD1431 or equivalent)
• 100% Ethanol (200-proof) for preparation of 70% ethanol by dilution with PCR-
grade water
• MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche Cat. No. 03730 964001)
• MagNA Pure LC Total Nucleic Acid Isolation Kit-Additional Lysis/Binding Buffer
(Roche Cat. No. 03246779001)
• TaqMan® Universal PCR Master Mix (Life Technologies, Cat. No. 4304437)
• TaqMan® Platinum® Taq DNA Polymerase (Life Technologies, Cat. No. 10966-034)
• Primers and probe fori7, tularensis Type A specific strains F4_pdpD targeting the
pathogenicity determinant protein D (obtained from a commercial supplier, Biosearch
Technologies or similar)
• Forward Primer (F4_pdpD_F) -
5' - TTG CTC CAG TAG CTG CAA GAT T -3'
• Reverse Primer (F4_pdpD_R) -
5' - CCA AGT GCT TGG TGG TGG TA -3'
• Probe (F4_pdpD_Pr) -
5'-6FAM- TGC TGC CGA GAT GTT TTC ATT ATT AAC TGA TGC -BHQ1-
3'
• Primers and probe fori7, tularensis Type A specific strains F5_pdpD targeting the
pathogenicity determinant protein D (obtained from a commercial supplier, Biosearch
Technologies or similar)
• Forward Primer (F5_pdpD_F) -
5'-GAG AC A TCA ATT AAA AGA AGC AAT ACC TT-3'
• Reverse Primer (F5_pdpD_R) -
5'-CCA AGA GTA CTA TTT CCG GTT GGT-3'
• Probe (F5_pdpD_Pr) -
5'-6FAM-AAA ATT CTG CTC AGC AGG ATT TTG ATT TGG TT-BHQ1-3'
• Bacto™ Brain Heart Infusion Broth Base (BD Biosciences Cat No. 237500)
• L-histidine (Sigma-Aldrich Cat. No. H8000-5G)
• Oxoid® Vitox Supplement (Thermo Fisher Scientific Cat. No. SR0090A)
• Remel™ Fildes Enrichment supplement (Thermo Fisher Scientific Cat. No. R45037)
6X Brain Heart Infusion (BHI) Broth with 20% (v/v) Vitox Supplement (final 2%
Vitox in IX), 60% (v/v) Fildes (final 10% Fildes in IX), and 1% (w/v) L-histidine
(BVFH)
1. Weigh 22.2 g Bacto™ Brain Heart Infusion Broth Base powder into 500 mL flask
or bottle.
2. Weigh 0.6 g L-histidine into the same 500 mL flask or bottle.
3. Add 28 mL MilliQ® H2O or equivalent.
4. Heat with frequent agitation and boil for 1 minute to completely dissolve the
powder.
5. Allow broth to cool down to room temperature under a BSC before storing at
4°C.
54
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6. Check pH and adjust by addition of NaOH pellets if pH is under 6, such that the
pH is 7.2 ± 0.2.
7. For addition of Oxoid® Vitox Supplement, reconstitute Vitox supplement in
buffer provided by vendor and add 12 mL of Vitox supplement to give a
concentration of 12% in the sterilized 6X BHI+histidine broth (2% when diluted
to IX).
8. For addition of Remel™ Fildes Enrichment supplement, add 60 mL Fildes to give
a concentration of 60% in the sterilized 6X BHI+histidine+Vitox broth (10%
when diluted to IX).
9. Test samples of the 6X BVFH product for sterility by incubating a 0.6 mL aliquot
into 5.4 mL buffer in a 50-mL conical tube and incubating at 37°C for 3 days.
Confirm sterility before use in the RV-PCR assay.
IX Phosphate-Buffered Saline (IX PBS)
Note: IX PBS may be preparedfrom 1 OX PBS Buffer or used as IX (pH 7.4; Teknova Cat. No.
P0261)
1. Add 100 mL 10X PBS buffer (Teknova Cat. No. P0195) to a 1-L flask or bottle.
2. Add 900 mL MilliQ® FhO or equivalent.
3. Sterilize 15 min at 15 psi and 121°C or through 0.2 micron 1-L disposable
filtration unit.
4. Store in refrigerator.
10% Bleach-pH amended (prepared daily)
• Prepare bleach solution by adding 1-part bleach (Ultra Clorox® Germicidal
bleach), 1-part acetic acid and 8 parts reagent-grade water as described below.
• 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 of water sample 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.
55
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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 (up to 4,500 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. Vortex mix the remaining 3 mL and the pellet.
g. Remove suspension (or combined suspension) from one tube with a sterile
5 mL pipet (recording the volume) and transfer to a corresponding sample
well of a 48-well plate.
2. Add concentrated growth medium (6X BVFH) and process for RV-PCR analysis.
a. Add 600 |iL of 6X BVFH to each well of the 48-well plate using a 1000
|iL pipettor (Final BVFH ~ IX). Mix the sample and medium well.
b. For each well, transfer 500 |iL from each sample well of the 48-well plate
and transfer to an appropriately labeled 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-lok®Ziploc bag and seal bag.
c. Incubate 48-well plate at 37°C on a shaker incubator at 180 rpm for 30
hour (h).
4. Process To aliquot for DNA Extraction-Purification
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 (see DNA Extraction/Purification
Procedure section).
5. At T30 (after 30-h incubation), transfer 500 |iL from each sample well to an
appropriately labeled 2-mL screw cap tube. Ensure that the T30 aliquot for each
sample is taken from the same well from which the To aliquot for the
corresponding sample was taken. This is a T30 aliquot for each sample.
Note: For post-decontamination, field samples (with potentially high
concentrations of dead F. tularensis cells), the incubation period may be extended
to 36 h.
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.
56
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6. Process the pellets from To and T30 aliquots by the DNA Extraction/Purification
Procedure below.
RV-PCR Analysis: DNA Extraction/Purification Procedure (Promega MagneSil® Blood
kit)
Note: To and T30 extractions can be completed separately.
1. Thaw To and T30 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 T30 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 T30 lysate
(containing 1 mL sample), hereafter referred to as "To and T30 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 T30 tubes.
7. Vortex each To and T30 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
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 T30 tube, 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.
57
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10. After adding lysis buffer to all the To and T30 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 T30 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 T30 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 T30 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 T30 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 T30 tube and
discard used tips in an appropriate waste container. Recap the tube. Repeat for all
To and T30 tubes.
16. 2nd Salt Wash: Repeat Salt Wash for all To and T30 tubes.
17. 1st Alcohol Wash: Uncap each To and T30 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 the To and T30 tubes, vortex each tube
for 5-10 seconds (low speed) and place on the magnetic stand. After all the To
and T30 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 T30 tube and
discard used tips in a sharps container. Recap the tube.
20. 2nd Alcohol Wash: Repeat Alcohol Wash for all To and T30 tubes.
21. 3rd Alcohol Wash: Repeat Alcohol Wash for all To and T30 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 T30 tubes and air dry for approximately 2 minutes.
23. Heat the open To and T30 tubes in the heat block at 80°C ± 2°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 T30 tube, and close
tube.
25. Vortex for 10 sec (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.
58
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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 T30 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. 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 T30 DNA extract tubes "referred to as To and T30 DNA extracts" at
4°C until PCR analysis (use photo-tray to transport 1.5 mL tubes in a rack).
Note: If PCR cannot be performed within 24 hours, store DNA extracts at -20°C.
RV-PCR Analysis: DNA Extraction/Purification Procedure (Roche MagNA Pure Compact
kit)
1. When ready to proceed with inactivation, thaw pellet and heat lyse sample tubes by
incubating at 70°C ± 2°C for 10 min. Allow sample tubes to cool briefly (2-3 min).
2. Add 300 [j,L lysis/binding buffer (MagNA Pure® LC Total Nucleic Acid Isolation Kit
Lysis/Binding Buffer-Refill; Roche, Cat. No. 03 246 779 001) to each sample tube and
close sample tube cap. Vortex on single tube vortexer for 5 sec at 1800-2000 rpm (VWR,
IK A Model MV1 or equivalent).
3. Add 300 [j,L phosphate-buffered saline (PBS; IX PBS, Teknova, Cat. No. P0261) to each
PC Tube.
4. Vortex tubes for 10 sec on single tube vortexer at 1800-2000 rpm (VWR, IKA Model
MV1 or equivalent).
5. Incubate tubes for 30 min at room temperature (in BSC). Every 5 min invert tubes 5
times to mix.
DNA Purification Protocol
Note: Run samples in batches of 8 on the instrument.
1. Adapt the Reagent Cartridge to room temperature (15 to 25°C) before use. The kit may
not work well at temperatures outside the recommended range.
2. Use the MagNA Pure® Compact Nucleic Acid Isolation Kit I and select the
DNA_Blood_external_lysis purification protocol (supplied with the MagNA Pure®
Compact instrument). The sample and elution volumes must be chosen from the software
menu.
3. Samples will be lysed and filtered manually using 0.2 micron Ultrafree-MC filter units
(Millipore Cat. No. UFC30GV0S) following manufacturer's instructions, outside the
59
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MagNA Pure® Compact instalment. Filtered lysates are then transferred to the instrument
and purification is carried out automatically. This procedure allows for physical
separation of the initial lysis/inactivation step(s) from the purification steps and enables
use of inactivated sample material on the MagNA Pure® Compact instrument (e.g., when
using potentially infectious sample material).
MagNA Pure® Compact Protocol Using the MagNA Pure Compact Nucleic Acid Isolation
Kit I
Isolate nucleic acids according to the protocol below:
4. Turn on the instrument; ensure that the Tube Rack is seated correctly in the instrument.
5. Remove the Elution Tube Rack from the instrument.
6. Click the Run button on the Main Menu Screen to access Sample Ordering Screen 1.
7. Follow the software-guided workflow.
8. Remove a prefilled Reagent Cartridge from the blister pack. Handle each Reagent
Cartridge as follows:
a. Always wear gloves when handling the cartridge.
b. Hold the cartridge only at the barcode imprinted area and the opposite side.
c. Avoid touching the sealing foil covering the cartridge wells.
d. Avoid touching the two single open wells and do not use them as handles.
e. Avoid any foam formation and let the fluid within the cartridge wells settle again
completely. If fluid remains under the sealing foil, knock the cartridge bottom
gently on a flat lab bench surface. This is especially important for well 1 which
contains a small volume of Proteinase K.
9. Check the cartridge integrity and filling volumes of the wells. Do not use cartridges that
have a different pattern of filling or that are damaged.
10. Scan the cartridge barcode using the barcode scanner supplied with the instrument.
11. With the two isolated wells pointing away from you, insert all the wells on the Reagent
Cartridge into the holes in the Cartridge Rack.
12. Use the guide slots on the rack to help position the cartridge.
13. Repeat the steps above for the desired numbers of samples (1 to 8).
14. Proceed to Sample Ordering Screen 2.
15. Select the appropriate purification protocol from the Protocol menu
(DNA_Blood_external_lysis).
16. Select the elution volume (100 (j,L).
17. Optional: Select the Internal Control Volume (0 |iL),
18. Insert the appropriate number of Tip Trays (one per sample) into the assigned position in
the instrument Tip Rack.
19. Check if the Tip Tray holds a disposable tip or piercing tool in each position. Do not use
tip trays that are not assembled accordingly.
20. Handle Tip Trays with care to prevent tips or piercing tool from falling out of the tray.
Should this happen, discard the respective tip tray and tips. Use the Tip Tray Kit to
replace missing Tip Trays.
21. Proceed to Sample Ordering Screen 3.
22. Scan the sample barcode from the primary sample tube or enter the sample ID.
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23. One at a time, uncap and arrange the Sample Tubes in row 1 of the Tube Rack. Make
sure the brim of the tubes seats solidly on the rack. Discard caps to waste.
24. Scan the bar codes of the Elution Tubes.
25. Place the Elution Tubes into the Elution Tube Rack. Make sure the brim of the tubes
seats solidly on the rack.
26. On the Confirmation Screen, check the information display.
27. If the information is correct, confirm it by touching the "Confirm Data" button, close the
front cover, and start the run.
28. After the purification run has ended, the Result Screen appears showing the result of the
isolation process for each channel.
29. The result will be PASS if the isolation run was completed without any warning or error.
30. The result will be FAIL if any interruption of the process or error occurred during the
run. For each FAIL result, the result screen will show a brief error or warning messages
to help you decide whether the error or warning can be ignored. Refer to the
troubleshooting section of the MagNA Pure® Compact Operator's Manual.
31. Close the Elution Tubes with the supplied tube caps and remove the Elution Tube Rack
or the Elution Tubes immediately after the end of the purification run.
32. If not proceeding directly to your downstream application, store DNA eluates at -20°C.
DNA is stable for at least 6 to 12 months if stored properly.
33. Optional: Start the automated liquid waste discard.
34. Always empty the MagNA Pure® Compact Waste Tank after every purification run.
35. Treat liquid waste as potentially infectious (depending on sample material), and
hazardous, since lysis buffers are present (see Safety Information in instrument manual
and/or SDS for the lysis buffer).
36. Store DNA extract tubes "referred to as To or T30 DNA extracts" at 4°C until PCR
analysis (use photo-tray to transport 1.5 mL tubes in a rack).
Note: If PCR cannot be performed within 24 hours, freeze DNA extracts at -20°C.
Cleanup Procedure
• Dispose of all biological materials (double bagged) in autoclave bags and sealed.
• Autoclave all waste materials.
• Decontaminate counters and all equipment with bleach (1 volume water and 9 volumes
commercial bleach) made fresh daily. Follow this with rinsing by 70% isopropanol
and/or by rinsing with deionized water.
Real-time PCR Analysis
1. Prepare PCR Mix according to the table below (PCR Mix for All Selected F. tularensis
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 T30 DNA extracts on same PCR plate.
61
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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
UNG
Incubation
AmpliTaq
Gold
Activation
PCR, 45 cycles
HOLD
HOLD
Denaturation
Annealing/Extension
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 F4 and F5 F. tularensis Real-time PCR Assays
Reagent
Volume (jiL)
Final
Concentration
TaqMan® 2X Universal Master Mix
12.5
IX
Platinum® Taq Polymerase
0.25
1.25 U
Forward primer, 10 |iM
0.5
0.20 |iM
Reverse primer, 10 |iM
0.5
0.20 |iM
Probe, 4 |xM
0.4
0.064 |iM
Molecular Biology Grade Water
5.85
N/A
Template DNA
5
Variable
TOTAL
25
RV-PCR Data Interpretation
Calculate an average Ct from the replicate reactions for To and T30 DNA extracts of each sample.
Subtract the average Ct of the T30 DNA extract from the average Ct of the To DNA extract. If
there is no Ct value for the To or T30 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 T30 (ACt) indicates a positive result suggesting the presence of viable F. tularensis cells in the
62
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sample. A ACt criterion of > 6 (an approximate two log difference in DNA concentration) and a
corresponding T30 Ct of < 39 was set. If an incubation time longer than 30 hours was used for
the RV-PCR, instead of T30, appropriate Tf (incubation time) shall be used (i.e., 36 h for post-
decontamination, field samples with high concentrations of dead F. tularensis 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 T30 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 because 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 because 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 are considered valid or if the PCR analyses must be
repeated.
Traditional culturing of diluted cell suspensions on Chocolate Agar (or other appropriate
media)
1. Inoculate Chocolate Agar plates with 100 |iL of each sample (each dilution is plated in
triplicate).
2. Using one Lazy-L™ cell spreader (Millipore Sigma, Burlington, MA) 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 Chocolate Agar plates at 37°C for 3 days.
5. Place sealed sample tubes in a secondary container (re-sealable bag); store tubes at 4°C.
6. After three days, confirm growth fori7, tularensis. Confirm that a subset of the colonies
is characterized as F. tularensis based on real-time PCR analysis using the F4 or F5 F.
tularensis-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)
63
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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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