EPA/600/R-10/156 | January 2011 | www.epa.gov/ord
United States
Environmental Protection
Agency
Development and Verification
of Rapid Viability Polymerase
Chain Reaction (RV-PCR)
Protocols for Bacillus anthracis
- For application to air filters,
water and surface samples
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R-10/156 | January 2011 | www.epa.gov/ord
LLNL-TR-424891
United States
Environmental Protection
Agency
Development and Verification
of Rapid Viability Polymerase
Chain Reaction (RV-PCR)
Protocols for Bacillus anthracis
- For application to air filters,
water and surface samples
Office of Research and Development 'Lawrence Livermore National Laboratory
"""National Homeland Security Research Center 7000 East Avenue, Livermore, CA 94550
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EPA
This document has been reviewed in accordance with EPA policy and approved for publication.
Mention of trade names or commercial products does not constitute endorsement or recommendation
for use. Note that approval does not signify that the contents necessarily reflect the views of the
Agency. Mention of trade names, products, or sen-ices does not convey EPA approval, endorsement,
or recommendation.
LLNL
This document was prepared as an account of work sponsored by an agency of the United States
government. Neither the United States government nor Lawrence Livermore National Security.
LLC. nor any of their employees makes any warranty, expressed or implied, or assumes any legal
liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus,
product, or process disclosed, or represents that its use would not infringe privately owned
rights. Reference herein to any specific commercial product, process, or sendee by trade name,
trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement.
recommendation, or favoring by the United States government or Lawrence Livermore National
Security, LLC. The views and opinions of authors expressed herein do not necessarily state or reflect
those of the United States government or Lawrence Livermore National Security. LLC, and shall not
be used for advertising or product endorsement purposes.
The work presented in this report was performed within a Quality Assurance Project Plan agreed
upon by the U.S. Environmental Protection Agency and LLNL.
Questions concerning this document or its application should be addressed to:
Sanjiv R. Shah, Ph.D.
National Homeland Security Research Center
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue. NW
USEPA-8801RR
Washington, DC 20460
(202) 564-9522
shah, sanj iv@epa.gov
If you have difficulty accessing these PDF documents, please contact Nickel.Katliyi@epa.gov or
McCall.Amelia@epa.gov for assistance.
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The mission of the U.S. Environmental Protection Agency (EPA) is to protect human health and
to safeguard the natural environment - the air, water, and land upon which life depends. The
series of 2001 terrorist attacks, including the anthrax bioterrorism incidents that resulted in human
casualties and public facility closures, prompted enhanced and expanded national safeguards.
Presidential directives identified EPA as the primary federal agency responsible for the protection
and decontamination of indoor-outdoor structures and water infrastructure vulnerable to chemical,
biological, or radiological (CBR) terror attacks. EPA's mission, to protect human health and the
environment, was thereby expanded to address critical needs related to homeland security.
The National Homeland Security Research Center (NHSRC) within the Office of Research and
Development (ORD) is EPA's hub for providing expertise on CBR agents, and for conducting and
reporting research to meet its homeland security mission needs. A focus of NHSRC's research is
to support the Environmental Response Laboratory Network (ERLN), a nationwide association of
federal, state, local, and commercial environmental laboratories, established by EPA. The ERLN
can be deployed in response to a large-scale environmental disaster to provide consistent analytical
capabilities, to offer increased capacity, and to produce quality data in a systematic and coordinated
manner. Preparedness against potential indoor or outdoor wide-area anthrax attacks is currently the
highest priority for the ERLN. To this end. NHSRC has developed and verified the Rapid Viability
PCR (RV-PCR) method for detection of live anthrax spores in environmental samples.
This report provides a detailed account of the development and verification of the RV-PCR method
for detection of live spores of Bacillus anthracis Ames in environmental sample matrices such as the
Bio Watch air filters, surface sampling wipes, and water.
NHSRC has made this publication available to assist in preparing for and recovering from disasters
involving anthrax spores contamination. This work specifically represents a very important step in
NHSRC's support for the ERLN. It is also key to the Agency's commitment to fulfill its homeland
security mission and its overall mission to protect human health and the environment.
Jonathan Herrmann, Director
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
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Disclaimers iii
Foreward
List of Figures ix
List of Tables x
List of Acronyms xv
Trademarked Products xvi
Executive Summary xvii
1.0 Introduction 1
2.0 Quality Assurance Project Plan
3.0 Materials and Methods
4.0 Results and Discussion 11
4.1 Assay Development 11
4.1.1. In sili co Analysis 11
4.1.2. Assay Optimization 11
4.1.3. Selectivity Study 15
4.2. Select Agent Laboratory Set Up 17
4.3. Rapid Viability -Poly unerase Chain Reaction (RV-PCR) Method Development 17
4.3.1. Development of Manual RV-PCR 18
4.3.2. Development of Semi-automated RV-PCR 21
4.3.3. Method Optimization 25
4.3.3.1. Improvement of Sample Mixing after Incubation 25
4.3.3.2. Shortening of the RV-PCR Method Endpoint for More Rapid Detection
of Bacillus anthracis Spores 26
4.3.4. Development of a TO Control Protocol 29
4.4. Single Laboratory Method Verification 34
4.4.1. Verification of the Manual RV-PCR Method 34
4.4.2. Verification of tlie Semi-Automated RV-PCR Method 39
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5.0 Conclusions 47
6.0 Acknowledgments 49
7.0 References 51
Appendix A: Manual RV-PCR Protocol 53
Appendix B: Semi-automated RV-PCR Protocol 59
Appendix C: Final Manual RV-PCR Protocol 65
Appendix D: Final Semi-automated RV-PCR Protocol 69
Appendix E: Lab Clean up Procedure, Buffers and Media 73
Appendix F: PCR Conditions 75
Appendix G: Consumables 77
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List of
Figure 1. Summary of the RV-PCR protocol steps 7
Figure 2. Magnetic rack used to process samples with the Promega DN A clean up kit 8
Figure 3. Summary of liquid handling steps performed during the RV-PCR protocol 9
Figu re 4. PCR efficiency of the three down-selected assay s for Ba Ames 15
Figure 5. Pictures of the EPA-dedicated Select Agent laboratory 17
Figure 6. Summary of Ct values obtained with the manual RV-PCR protocol on both
clean and dirty sample types 19
Figure 7. Janus robotic platform and custom HEPA-Ollered enclosure in the Select
Agent laboratory 22
Figure 8. Summary of Ct values obtained with manual and semi-automated RV-PCR
protocols on clean samples 25
Figure 9. Summary of Ct values obtained with the semi-automated RV-PCR protocol on clean
samples with and without vortexing filter cups 26
Figure 10. Optimized RV-PCR method overview 29
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List of
Table 1. Comparison of Rapid Viability-Polymerase Chain Reaction and Plating Methods I
Table 2. Summary of All Bacillus anthracis Signatures Analyzed in Silico 11
Table 3. List of 10 Bacillus anthracis Assays Selected After in Silico Analysis 11
Table 4. Measurement of Bacillus anthracis Ames DNA Concentration by Nanodrop™
UV Spectrometer and Qubit® Fluorimeter based methods 12
Table 5. Initial Screening of Bacillus anthracis Chromosome Assays BC1, BC2 and BC3 12
Table 6. Initial Screening of Bacillus anthracis pXOl Plasmid Assays BP11, BP12, BP13
andEPA-1 12
Table 7. Initial Screening of Bacillus anthracis pXO2 Plasmid Assays BP21, BP22, andEPA-2.... 12
Table 8. Nucleotidc Sequences and Amplicon Size for Selected Bacillus anthracis
RV-PCR Assays 13
Table 9. Optimization of Primer and Probe Concentration for the BC3 Assay 13
Table 10. PCR Mix for pXO2 (EPA-2) Primer/Probe Set 13
Table 11. PCR Mix for Chromosome (BC3) andpXOl (EPA-1) Primer/Probe Sets 14
Table 12. PCR Thermal Cycling Conditions for All 3 Primer/Probe Sets 14
Table 13. Performance of Optimized BC3, EPA-1, and EPA-2 PCR Assays Tested
With Bacillus anthracis Ames DNA 14
Table 14. Change in average Ct value for Bacillus anthracis Ames Assays with BHI
Medium in the PCR, Compared to Water 15
Table 15. Change in average Ct value for Bacillus anthracis Ames Assays with
lysed culture of Bacillus atrophaeus (109 cells/mL) in the PCR, Compared to Water 15
Table 16. PCR Assay Selectivity Study Using a Panel of Bacillus anthracis Strain DNA 16
Table 17. PCR Assay Selectivity Study Using a Panel of Near-Neighbor Bacillus species DNA 16
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Table 18. Manual RV-PCR at T16 on Clean Wipes Spiked With 3 Spore Levels 18
Table 19. Manual RV-PCR at T16 on Clean and Dirt}' Wipes Spiked With 2 Spore Levels 18
Table 20. Manual RV-PCR at T16 on Clean Filters Spiked With 3 Spore Levels 18
Table 21. Manual RV-PCR at T16 on Dirty Air Filters Spiked With 3 Spore Levels 19
Table 22. Manual RV-PCR at T16 on Clean and Dirty Water Spiked With 3 Spore Levels 19
Table 23. Manual RV-PCR at T16 on Clean Wipes Spiked With 2 Spore Levels in the
Presence of B. globigii and P. aentginosa Background 20
Table 24. Manual RV-PCR at T16 on Clean Filters Spiked With 2 Spore Levels in the
Presence of 5. globigii and/! aeruginosaBackground 20
Table 25. Manual RV-PCR at T16 on Clean Water Spiked With 2 Spore Levels in the
Presence ofB. globigii sad P. aeruginosa Background 21
Table 26. Manual RV-PCR at T16 on Clean Wipe, Air Filter and Water Samples Spiked
With 2 Live Spore Levels in the Presence of Heat-Killed Bacillus anthracis Ames Spores 21
Table 27. Semi-automated RV-PCR at T16 on Clean Wipe, Air Filter and Water Samples
Spiked With 2 Spore Levels 22
Table 28. Semi-automated RV-PCR at T16 on Dirty Wipe. Air Filter and Water Samples Spiked
With 2 Spore Levels '. 23
Table 29. Semi-automated RV-PCR at T16 on Clean Wipe, Air Filter and Water Samples Spiked
With 2 Spore Levels in the Presence of Heat-Killed Bacillus anthracis Spore Background 23
Table 30. Semi-automated RV-PCR at T16 on Clean Wipes Spiked With 2 Spore Levels in
the Presence of B. globigii and P. aeruginosa Background 24
Table 31. Semi-automated RV-PCR at T16 on Clean Filters with 2 Live Spore Levels in the
Presence of B. globigii and P. aeruginosa Background 24
Table 32. Semi-automated RV-PCR at Tl 6 on Clean Water Spiked With 2 Spore Levels in the
Presence of B. globigii and P. aeruginosa Background 24
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Table 33. Semi-automated RV-PCR at T16 on Clean Wipe. Air Filter and Water Samples
Spiked with 36 Bacillus anthracis Ames Spores 25
Table 34. Manual RV-PCR at T9 on Heat-Lysed Extracts of Clean Wipe, Air Filter and
Water Samples Spiked With 2 Spore Levels 26
Table 35. Manual RV-PCR at T9 on Clean Wipe, Air Filter and Water Samples Spiked With
2 Spore Levels and Processed with the DN A Extraction and Purification Protocol 27
Table 36. Manual RV-PCR at T9 on Dirty Wipe, Air Filter and Water Samples Spiked With
2 Spore Levels and Processed with the DNA Extraction and Purification Protocol 27
Table 37. Manual RV-PCR After Heat Lysis at TO on Wipes Spiked with 28 Bacillus anthracis
Spores 28
Table 38. Manual RV-PCR at T8 on Wipes Spiked With 28 Bacillus anthracis Spores 28
Table 39. Manual RV-PCR at TO on Wipes Processed with DNA Extraction and Purification of
on 1 mL Aliquots 30
Table 40. Manual RV-PCR on Wipes Processed with DNA Extraction and Purification on 1 mL
Aliquot at TO Followed by 1:10 Dilution of the Extracted Samples 30
Table 41. Experiment Design for Aliquot Equivalency between TO and T9 Samples 31
Table 42. Manual RV-PCR on Wipes Processed with DNA Extraction and Purification at TO
(Table 41 Column 1)
Table 43. Manual RV-PCR at T9 on Wipes with Heat Lysis 32
Table 44. Manual RV-PCR at T9 on Wipes Processed with DNA Extraction and Purification
Followed by 1:10 Dilution of Extracted Samples 32
Table 45. Manual RV-PCR at T9 on 1:17 Diluted Purified DNA from Wipes
(Table 41 Column 3) 33
Table 46. Ct Values Obtained with Extracted B. glohigii DNA Dilution Series 33
Table 47. Ct values for 1 mL Samples Spiked with 103 Bacillus anthracis CFU/mL and
B. glohigii DNA, and Processed with DNA Extraction and Purification 34
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Table 48. Summary of Aliquot Equivalency between TO Controls and T9 Samples for
the Processing of Real-World Samples 34
Table 49. Average Ct Values for Manual RV-PCR at T9 on Clean Wipes with 2 Spore Levels 35
Table 50. Average Ct Values for Manual RV-PCR at T9 on Clean Filters with 2 Spore Levels 35
Table 51. Average Ct Values for Manual RV-PCR at T9 on Clean Water with 2 Spore Levels 35
Table 52. Average Ct Values for Manual RV-PCR at T9 on Dirt}' Wipes with 2 Spore Levels 36
Table 53. Average Ct Values for Manual RV-PCR at T9 on Dirt}' Filters with 2 Spore Levels 36
Table 54. Average Ct Values for Manual RV-PCR at T9 on Dirty Water with 2 Spore Levels 36
Table 55. Average Ct Values for Manual RV-PCR at T9 on Clean Wipes with 2 Spore Levels
in the Presence of Heat-Killed Bacillus anthracis Spore Background 37
Table 56. Average Ct Values for Manual RV-PCR at T9 on Clean Filters with 2 Spore Levels
in the Presence of Heat-Killed Bacillus anthracis Spore Background 37
Table 57. Average Ct Values for Manual RV-PCR at T9 on Clean Water with 2 Spore Levels
in the Presence of Heat-Killed Bacillus anthracis Spore Background 38
Table 58. Average Ct Values for Manual RV-PCR at T9 on Clean Wipes with 2 Spore Levels
in the Presence of live/-! aeruginosa andli. globigii Background 38
Table 59. Average Ct Values for Manual RV-PCR at T9 on Clean Filters with 2 Spore Levels
in the Presence of live P. aeruginosa and B. globigii Background 39
Table 60. Average Ct Values for Manual RV-PCR at T9 on Clean Water with 2 Spore Levels
in the Presence of live P. aeruginosa and B. globigii Background 39
Table 61. Average Ct Values for Semi-automated RV-PCR at T9 on Clean Wipes with
2 Spore Levels 40
Table 62. Average Ct Values for Semi-automated RV-PCR at T9 on Clean Filters with
2 Spore Levels 40
Table 63. Average Ct Values for Semi-automated RV-PCR at T9 on Clean Water with
2 Spore Levels 40
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Table 64. Spore Recovery Experiment from Filter Cup Aliquots at TO and T9 41
Table 65. Average Ct Values for Semi-automated RV-PCR at T9 on Dirty Wipes with
2 Spore Levels 42
Table 66. Average Ct Values for Semi-automated RV-PCR at T9 on Dirt}' Filters
with 2 Spore Levels 42
Table 67. Average Ct Values for Semi-automated RV-PCR at T9 on Dirty Water with
2 Spore Levels 42
Table 68. Average Ct Values for Semi-automated RV-PCR at T9 on Clean Wipes with
2 Spore Levels in the Presence of Heat-Killed Bacillus anthmcis Spore Background 43
Table 69. Average Ct Values for Semi-automated RV-PCR at T9 on Clean Filters with 2 Spore
Levels in the Presence of Heat-Killed Bacillus anthracis Spore Background 43
Table 70. Average Ct Values for Semi-automated RV-PCR at T9 on Clean Water with
2 Spore Levels in the Presence of Heat-Killed Bacillus anthracis Spore Background 43
Table 71. Average Ct Values for Semi-automated RV-PCR at T9 on Clean Wipes with 2 Spore
Levels in the Presence of P. aeruginosa and B. globigii Background 44
Table 72. Average Ct Values for Semi-automated RV-PCR at T9 on Clean Filters with 2 Spore
Levels in the Presence of P. aeruginosa and B. glohigii Background 44
Table 73. Average Ct Values for Semi-automated RV-PCR at T9 on Clean Water with
2 Spore Levels in the Presence off! aeruginosa and 5. glohigii Background 44
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List
ABI: Applied BioSystems, Inc.
ATCC: American Type Culture Collection
AZ: Arizona
Ba: Bacillus anthracis
Bg: Bacillus atrophaens subsp. globigii
BHI: Brain Heart Infusion
BHQ: Black Hole Quencher
bp: Base Pairs
BSC: Biosafety cabinet
CDC: Centers for Disease Control and Prevention
CFU: Colony Forming Unit
CT/Ct: Cycle Threshold
DHS: Department of Homeland Security
DNA: Deoxyribonucleic acid
DTMA: Defense Threat Reduction Agency
ERLN: Environmental Response Laboratory Network
EPA: U. S. Environmental Protection Agency
FAM: 6-carboxy-fluorescein
fg: femtogram
BDPLC: High Performance Liquid Chromatography
HSAMPA: Homeland Security Advanced Research Project Agency
IBRD: Interagency Biological Restoration Demonstration
ISO: International Organization for Standardization
L: Liter
LLNL: Lawrence Livermore National Laboratory
LOD: Limit of detection
LRN: Laboratory Response Network
Mb: Megabases
mg: Milligram
|iL: Microliter
MPN : Most probable number
NBBT: Neutralization Butterfield's Buffer
NUT: Non-detected
ng: Nanogram
NHSRC: National Homeland Security Research Center
PCR: Polymerase Chain Reaction
Pa: Pseudomonas aeruginosa
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pg: Picogram
PPE: Personal Protection Equipment
PSAA: Public Safety Actionable Assays Program
PSU: Portable Sampling Unit
QAPP: Quality Assurance Project Plan
q-PCR: Quantitative PCR
UNA: Ribonucleic acid
rpm: Rotations per minute
RV: Rapid Viability
RV-PCR: Rapid Viability- Polymerase Chain Reaction
SOP: Standard Operating Procedure
TO: Time 0, prior to incubation
T9: After 9 hr of incubation
T16: After 16 hr of incubation
TE: Tris-etliylene diamine tetra acetic acid
UNG: Uracil-N-Glycosilase
UV: Ultra Violet
WA: Work Assignment
Trademark
ABI GOLD™'
AB Applied BioSystems™
ATCC™
AmpliTaq™
Cole Farmer*
Epicentre*
Excel*
GenBank*
Invitrogen*
Life Technologies™
MagneSil* Blood Genomic
Microsoft*
Millipore*
MyCycler™
Nanodrop™
PicoGreen®
Quant-iT™
Quhit*
Qubit™
TaqMan™
Holder
Life Technologies
Life Technologies
American Type Culture Collection
ABI
Cole Farmer
Biotechnologies Inc.
Microsoft Corp.
U.S. Department of Health
and Human Services
Life Technologies
Life Technologies
Promega
Microsoft Corp.
Millipore Corp.
Bio-Rad Inc.
Thermo Scientific
Life Technologies
Life Technologies
Life Technologies
Life Technologies
Life Technologies
Location
Carlsbad, CA
Carlsbad, CA
Manassas, VA
Carlsbad, CA
Vemon Hills, IL
Madison, WI
Redmond, WA
Bethesda, MD
Carlsbad, CA
Carlsbad, CA
Madison, WI
Redmond, WA
Billerica, MA
Hercules, CA
Wilmington, DE
Carlsbad, CA
Carlsbad, CA
Carlsbad, CA
Carlsbad, CA
Carlsbad, CA
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There has been a critical need for the sophisticated
analytical tools necessary to rapidly detect and identify,
or rale out, live Bacillus anthracis, the inicrobial agent
for anthrax, during a bioterrorism event. Under an
interagency agreement between the U.S. Environmental
Protection Agency (EPA) and the U.S. Department
of Energy, and funding by EPA's National Homeland
Security Research Center (NHSRC), a cooperative
project was undertaken to provide these tools. The
Lawrence Livermore National Laboratory (LLNL) of
the Department of Energy and the National Homeland
Security Research Center spearheaded this project to
develop the rapid viability polymerase chain reaction
(RV-PCR) method for the detection and identification
of viable Bacillus anthracis spores in environmental
samples.
The RV-PCR method is aimed at serving the needs
of the Environmental Response Laboratory Network
(ERLN). The ERLN was established by EPA's Office
of Emergency Management to analyze environmental
samples during any intentional or natural contamination
event of national significance in order to both assess the
extent of contamination and evaluate decontamination
efficacy. The network, established to respond to
biological attacks in addition to chemical and
radiological attacks, would respond to any biological
attack by conducting sample analysis to determine
if facilities and areas have been restored to ambient
conditions. Validated, rapid viability test protocols
are needed as part of the ERLN capabilities to ensure
public safety and to help mitigate impacts due to facility
closures following a biological agent release. This
critical need for rapid analytical results was highlighted
during the response to the 2001. anthrax attacks in which
the clearance sampling and analysis required prior to
facilities re-opening took an excessive amount of time.
The focus of this effort was to develop real time PCR
assays for virulent Bacillus anthracis. In addition,
the work was to develop and verify manual and
semi-automated RV-PCR methods for detection and
identification of viable Bacillus anthracis Ames spores
in air filters, wipes, and water. Challenges administered
during testing included high populations of non-target
micro-organisms such as Bacillus airophaeus subspecies
globigii and Pseudomonas aeniginosa, high populations
of heat-killed Bacillus anthracis spores, and high
loadings of debris. Criteria for assessment of the PCR
methodology included limits of detection, accuracy with
plating, absence of PCR and growth inhibition, and turn-
around time for results.
Signatures for Bacillus anthracis were ranked and
selected using in silica analysis for signature specificity
against all available sequences in GenBank® (U.S.
National Institutes of Health's genetic sequence
database), virulence gene association, availability of
prior assay screening data and amplicon characteristics.
Signatures from multiple sources were considered,
including the Department of Homeland Security Public
Safety Actionable Assays Program, the Homeland
Security Advanced Research Project Agency program,
and EPA's NHSRC. Published assays as well as new
signatures developed for this project were also evaluated.
The output of this analysis was a computational
prediction of virulent Bacillus anthracis strain detection
for 44 candidate assays ranked for predicted selectivity,
amplicon size, and gene target. Ten assays (3 for the
chromosome, 4 for the pXOl plasmid and 3 for the
pXO2 plasmid) were selected based on the in silica
analysis and then optimized for real-time PCR. Three
assays (1 for the chromosome, 1 for the pXOl plasmid
and 1 for the pXO2 plasmid) were selected for RV-PCR
based on sensitivity, selectivity and robustness in the
presence of growth medium and cell debris.
Rapid viability protocols, initially developed for the
Interagency Biological Restoration Demonstration
Program and funded under the Department of Homeland
Security and the Department of Defense's Defense
Threat Reduction Agency, were leveraged and optimized
for Bacillus anthracis Ames using the selected assays.
The method endpoint was shortened from its original
16 lir to 9 hr, by performing a magnetic bead-based
DNA extraction and purification procedure prior to PCR
analysis. Using this improved method, the total analysis
time from start to finish for a batch of 24 samples is
reduced from 24 hr to 15 hr. For each subsequent batch
of 24 samples, only 3-4 additional hours are required.
Therefore, with adequate resources, it is possible to
analyze hundreds of samples per day.
Single laboratory verification of both manual and semi-
automated versions of this optimized method showed
limits of detection at the level of 10 spores per sample,
both with and without debris, for all three sample
types (clean laboratory water samples had a volume of
20 mL, wipes were 2 x 2" squares of rayon/polyester
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gauze, and air filters were 47 mm diameter discs of
hydrophobia polytetrafluoroethylene membranes).
Live Bacillus cmlhracis Ames spores were consistently
detected at the 10 spore level for both manual and semi-
automated methods in heat-killed Bacillus anthracis
spore backgrounds of 106 colony forming units per
sample, and combined non-target backgrounds of 103
live Bacillus atrophaetis subspecies globigii and 106 live
Pseudomonas aeruginosa. Follow on work will further
explore Hie relationship between limit of detection
and incubation time for clean wipe samples. These
experiments will be used to evaluate whether the method
endpoint could be further reduced when relatively clean
samples such as wipes collected from clean indoor
locations are processed.
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1.0
Introduction
To protect human health and ensure that the
environment and facilities are restored to ambient
conditions following a biothreat agent release, the U.S.
Environmental Protection Agency (EPA) needs rapid
viability testing methods to evaluate contaminants.
In the event of a biothreat agent release, hundreds to
thousands of samples of diverse types (aerosol, surface
and environmental) could need to be rapidly processed
in order to both characterize the extent of contamination
and determine the efficacy of remediation activities.
Decision-makers could also need rapid results for
re-mobilizing disinfection equipment in the case of
incomplete decontamination and for re-opening facilities
and areas based on results from clearance sampling.
Current viability test methods are too labor- and time-
intensive to be able to meet the need for rapid analysis.
Typically, with current methods, only 30-40 samples
are processed each day and the confirmed results are
obtained days later.
The EPA's Office of Emergency Management within
the Office of Solid, Waste, and Emergency Response
has established a network of laboratories to analyze
environmental samples, the Environmental Response
Laboratory Network (ERLN). The network was created
to respond to biological, chemical and radiological
attacks. The network would respond to a biological
attack by conducting sample analysis to determine
if facilities and areas have been restored to ambient
conditions. Validated, rapid viability test protocols
are needed as part of the ERLN capabilities to ensure
public safety and to help mitigate impacts that are due
to facility closures following a biothreat agent release.
This critical need was highlighted during the response
to the 2001. anthrax attacks in which clearance sampling
and analysis required excessive time prior to facilities
re-opening. Therefore, to establish a research project in
direct support of the ERLN, the EPA Office of Research
and Development's National Homeland Security
Research Center (NHSRC) initiated and entered into an
Interagency Agreement with the Lawrence Livermore
National Laboratory (LLNL) of the Department of
Energy (DOE). Under that agreement, the LLNL was
entrusted to develop and verify a rapid viability test
method for anthrax spores in various environmental
sample matrices. The NHSRC technical lead for this
project was Dr. Sanjiv R. Shah.
LLNL has leveraged the useful features of real-time
polymerase chain reaction (PCR) and expanded its
capabilities by conducting PCR analysis pre- and post-
incubation and using the change in the PCR cycle
threshold (Ct) value as an indicator for the presence
of viable spores or cells. The approach referred to
as "'rapid viability" (RV)-PCR uses high throughput
sample processing via commercial automation in
combination with 96-well real-time PCR to analyze
hundreds of surface samples per day (the advantages
of the RV-PCR method over the standard plating
method are summarized in Table 1). RV-PCR protocols
allow detection of low numbers of viable spores in
the presence of environmental backgrounds and high
populations of non-target microorganisms or dead
target spores. Under joint Department of Homeland
Security (DHS) and the Department of Defense's
Defense Threat Reduction Agency (DTRA) funding,
hundreds of samples spiked with Bacillus anthracis
(Ba) surrogates were processed using RV-PCR protocols
and demonstrating high throughput analysis and similar
detection limits and accuracy as traditional viability
analysis.1"2
Table 1. Comparison of Rapid Viability-Polyraerase
Chain Reaction and Plating Methods
Plating Method
(Gold Standard)
Direct method
(visible colonies)
Quantitative technique down
to 100 eel Is/ml,
Separate PCR
confirmation is needed
Live non-target backgrounds
may interfere with plate
counts
Method is labor intensive
(manual) and low throughput
(30-40 samples/lab/day)
Method has logistic
burdens (large
lab space required to
accommodate many BSCs
and incubators)
Time for results ~ 1 8
hr without confirmation
(confirmatory tests require
additional time and labor and
may take days)
RV-PCR Method
Indirect method (Ct values)
LOD < 10 cells/mL; method
is quantitative when combined
with MPN
PCR confirmation is built in the
method
Live non-target backgrounds do
not interfere with PCR
Method can be automated
and high throughput (100s of
samples/lab/day)
~ 96 filter cups can be
incubated in one standard
shaker incubator and processed
with one robot
Time for results < 15 hr
including confirmation
Acronyms: BSC, biosafety cabinet:
of detection; MPN, most probable
reaction; RV. rapid viability
:; Ct, cycle threshold; LOD, limit
number; PCR, polymerase chain
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The focus of the NHSRC-funded effort was to develop
real time PCR assays for virulent Ba and to develop
and verify manual and semi-automated RV-PCR
methods for virulent Ba Ames spores in water, wipes
and air filters. (These sample types were primarily
chosen based on their diversity (surface, air and water
samples) and on the availability of starting protocols
developed for the Interagency Biological Restoration
Demonstration (IBRD) program under joint DHS-
DTRA funding.) Challenges administered during testing
included high populations of non-target micro-organisms
such as Bacillus atrophaeus subsp. globigii (Bg) and
Pseudomonas aeruginosa (Pa) to evaluate the selectivity
of the method, high populations of heat-killed Ba spores
to evaluate decontamination scenarios, and high loadings
of debris to evaluate any PCR and/or growth inhibition
from environmental chemical and biological materials.
Criteria for assessment of PCR methodology included
limits of detection, accuracy with plating, absence of
PCR and growth inhibition, and turn-around time for
results. The current effort only developed and verified
the RV-PCR method for qualitative analysis of samples.
It should be noted that Hie work presented in this
report, did not focus on sampling methods and that an
exhaustive list of potential growth inhibitors and/or PCR
inhibitors was not assessed. Although dirt was added
to wipes, no actual surface wiping was performed to
test interferences from chemicals and/or debris from
sampled surfaces. Although air filter samples were
randomly collected from both subway and outdoor
locations, seasonal variations such as high versus low
pollen levels or high versus low air pollution levels were
not systematically tested. Although water samples were
spiked with humic acid and ferrous sulfate, which are
known PCR inhibitors, no environmental water samples
were tested in this study. Finally, any interference of the
decontamination method (fumigation, foam) with the
RV-PCR method will need to be tested. Typical effects
of decontamination are delayed germination and growth
and PCR inhibition. Such experiments were outside the
scope of this study since virulent Ba was used.
-------
2.0
Quality Assurance Project Plan
A quality assurance project plan (QAPP) was approved
by EPA before the experimental work started.3 All work
reported in this report was performed in accordance with
the QAPP.
Compliance with Centers for Disease Control (CDC)
and DOE safety and security policies was checked by
the project principal investigator in monthly laboratory
inspections. Quarterly laboratory inspections were
also conducted by the LLNL responsible official and
LLNL DOE representative. These quarterly inspections
included a review of laboratory cleanliness; the
certification of laboratory equipment including biosafety
cabinet, robotic enclosure and autoclave; a review of
the waste handling; an inventor)' of select agents; and
a review of personnel training and vaccination. EPA
program managers also toured our laboratory on multiple
occasions to ensure that work conducted met quality
assurance metrics. The ABI (Applied BioSystems™)
cycler was calibrated every 6 months, pipcttors were
inspected and calibrated by the vendor annually,
laboratory swipes were performed twice a year (the
second set of swipes were taken and analyzed just before
proceeding with single laboratory verification) and were
always negative for B. anthracis. The refrigerators and
freezers used are connected to an alann system, to ensure
storage conditions remain within acceptable ranges. In
addition, a temperature recording chart is inserted in the
refrigerator where stocks are maintained and checked
weekly. Thermometers are also placed in each incubator
to provide additional temperature control. During the
course of the experiment, B. anthracis Ames extracted
DNA standards were analyzed on every PCR plate, as
described in the Materials and Methods section. Plating
of the spiking spore suspension was conducted for each
experiment.
Data was reported and discussed with EPA's technical
lead monthly. Results obtained on the project were
also presented in front of the LLNL directorate review
committee, which is an external committee.
-------
-------
3.0
Materials and Methods
Detailed protocols for both manual and semi-automated
RV-PCR methods are provided in Appendices A and
B, respectively, as well as details of PCR conditions
(Appendix F), buffer and media preparation (Appendix
E), and consumables information (Appendix G).
All work presented in this report, including real-
time PCR assay evaluation and RV-PCR method
development, was conducted using the pathogenic
Bacillus anthracis Ames strain. This strain belongs to
the LLNL strain collection and has been verified by
performing real-time PCR analysis on genomic DNA
using primers and probes specific to the Ba chromosome.
and pXOl and pXO2 plasmids. The Ba Ames strain
was grown in Brain Heart Infusion (BHI) medium and
on BHI agar plates. Spore stocks were stored in a 70%
water and 30% etlianol solution at 4 °C.
B.
Ba was streaked for growth onto BHI agar and incubated
overnight at 36°C. The organism was then streaked
and incubated a second time for isolation. A 108 cells/
mL suspension of the 24 hr growth was prepared in
phosphate buffer (25mM KH2PO4, pH 7.2), plated
onto soil extract beef peptone agar and incubated at
36°C until 99% sporulation was achieved. Plates were
then scraped and rinsed using sterile water and a cell
scraper (the content of each plate was transferred to a
50 mL centrifuge tube in a total of 30 mL of water). The
spore preparation was cleaned using vortexing (2 min).
centrifugation (4000 rpmfor 15 min), removal of the
supernatant and addition of sterile water. This cleanup
procedure was repeated 4 times. Twenty milliliter of
a 1:1 (ethanol:water) solution were then added to the
centrifuge tubes, which were vortexed for 2 min to
re-suspend the spore pellets. Tubes were then placed
on a shaker platform for 1 hr at 80 rpm. After this step.
the spore suspension was washed again 7 consecutive
times using the vortexing. centrifugation and supernatant
exchange technique described above. The suspension
titer after these washing steps was 109 colony forming
units (CFU)/mL, as measured by plating. The fraction of
dead spores, measured by microscopy, was < 1%. The
final spore re-suspension was performed using a mixture
of 70% water and 30% ethanol in order to generate a
spore stock for storage at 4°C.
in
Three sample types were used in this study, including 2
x 2" wipes (Kendall; catalogue number 8042, 50% rayon
and 50% polyester gauze), 47 mm diameter air filters
(Millipore, catalogue number FSLW04700, hydrophobic
polytetrafluoroethylene membranes) and 20 mL water
samples (Milli-Q™-filtered water. pH 7.0.). These sample
types were primarily chosen based on their diversity
(surface, air and water samples) and on the availability
of starting protocols developed for the IBRD Program
under joint DHS-DTRA funding.
of dirty
The well-characterized Arizona Fine Test Dust (Powder
Technology Inc., Burnsville, MM) was used for this
study. The material consists of Arizona sand including
Arizona Road Dust, Arizona Silica, AC Fine and AC
Coarse Test Dusts, SAE Fine and Coarse Test Dusts,
J726 Test Dusts. ISO (International Organization for
Standardization) Ultrafine, ISO Fine, ISO Medium and
ISO Coarse Test Dusts, and MIL STD 810 Blowing
Dust.4 Analysis of chemical composition performed by
the manufacturer indicates that the material consists
of: SiO2 (68 to 76%), ALO, (10 to 15%), Fe2O3 (2 to
5%),Na,O (2 to 4%), CaO"(2 to 5%), MgO (1 to 2%),
TiO, (0.5 to 1.0%), and K2O (2 to 5%). Microbial
characterization of the test dust performed by the Centers
for Disease Control and Prevention (CDC) found 39
morphologically distinct colony types including Bacillus
cereus. Bacillus lichen/omits, Bacillus mycoides,
Bacillus endophyticus. actinomycetes, molds, yeast
micrococcus and streptomyces.5 A 0.5 mg/mL test dust
slurry stock was prepared by weighing 10 g of test dust
in a conical tube, adding 20 mL of deionized water,
and vortexing at high speed for 20 min. Five hundred
microliters of slurry were added to wipes (250 mg of
dust) as a challenge.
of dirty air
Air filters collected from portable air sampling
units belonging to the Bio Watch network (a DHS
environmental monitoring program) were used as
challenges for this study. Some filters came from
outdoor locations such as Houston, Texas, and others
came from indoor locations, such as a subway station in
Boston, Massachusetts. The goal of this challenge was
to assess the presence of growth and/or PCR inhibition
from aerosols present in both indoor and outdoor urban
-------
environments (including pollen, dust, dirt and chemical
and biological materials).
Preparation of chemically
The water used in this study was filtered through a
Milli-Q™ water system (Millipore. MA). Challenge
samples were prepared by adding ferrous sulfate and
humic acid at levels of 10 mg/L, which are known PCR
inhibitors.
Addition of heat-killed Ba
background
A stock of Ba Ames spores (106 CFU/mL confirmed by
plate counts) was killed by autoclaving three times at
126 °C and 15 psi for 30 min. Six 100 joL aliquots were
cultured on solid BHI medium and incubated for 48 hr
to confirm non-viability of the stock. One milliliter of
the heat-killed spore stock solution (106 CPU/sample)
was added to each sample type as a challenge. The goal
of this challenge was to simulate a decontamination
scenario in which low levels of live spores would need
to be detected in a high level of dead spores killed by the
decontamination method.
Addition of live non-target
A stock of Bacillus atrophaeus subspecies glohigii
(American Type Culture Collection [ATCC™] No. 9372)
was plated to confirm its spore concentration of 104
CFU/mL. One hundred microliters (103 spore level)
were then inoculated on each sample as a live challenge.
Pseudomonas aemginosa (ATCC No. 10145) cells were
grown overnight in a flask and diluted to a concentration
of 107 cells/mL using optical density measurements at
620 run to assess the cell concentration. One hundred
microliters (106 cell level) of this diluted culture were
inoculated on each sample. The final live background
for each sample was a combination of 103 Bg spores and
106 Pa cells. The goal of this challenge was to assess the
specificity of the RV-PCR method, as well as any growth
issues due to competition for nutrients.
with Ba
Prior to each RV-PCR experiment, the original Ba spore
stock (10s CFU/mL) was vortexed on a platform vortexer
for 20 min. Successive 10-fold dilutions were prepared
in phosphate buffer [25 inM KH2PO4, pH 7.4J, down to
102 CFU/mL. Three replicates of the last 2 dilutions were
cultured on agar plates following the traditional viability'
protocol below. Typically, 100 ^L of the 102 CFU/mL "
dilution and 50 p,L of the 103 CFU/mL- dilution were
plated in triplicate, in order to determine the inoculation
levels in terms of CPU/sample; this is why inoculation
levels slightly differed from experiment to experiment.
Typical error bars on the CFU/sample represent < 5% of
the mean value. Water, air filter and wipe samples were
inoculated using 100 joL of the 102 CFU/mL suspension
(10 spore level) and 100 ^L of the 103 CFU/mL
suspension (100 spore level). The targeted levels for this
study were the 10 spore level (1 to 99 CFU/sample) and
Hie 100 spore level (100 to 999 CFU/sample). Although
the goal was to test the lowest spore numbers for each
level, variability with pipetting, vortexing, and surface
binding of the spores to stock tubes generated slightly
different CFU values for each experiment, which were
quantified by systematic plating of the spiking solutions.
Traditional viability
For traditional viability analysis, 2 to 3 successive ten-
fold sample dilutions were cultured on BHI agar and
incubated overnight at 30 °C. From each dilution, three
100 |jJL aliquots were plated for spore count accuracy.
Colony counts were obtained the next day and corrected
for dilution in order to determine Hie number of viable
spores spiked in the samples.
Rapid-Viability PCR
The experimental protocol outline, as well as pictures
of the equipment used, are provided in Figure 1 and
detailed protocols are provided in Appendices A and B.
Briefly, samples were placed in 30 mL conical tubes and
spiked with Ba Ames spores as described above. A mesh
support was used to maintain wipe and air filter samples
in place. Twenty milliliters of extraction buffer (70%
of 0.25 mM KH,PO/0.1% Twecn 80 [pH 7.2] and 30%
ethanol; final pH ~9.5) was added to each tube (for wipes
and filters) and the tubes were vortexed for 20 min on
a platform vortexer to remove spores from the sample
matrix. Thirteen milliliters were then transferred from
each sample to a filter cup. and spores in the extraction
buffer were collected on a 0.45 jam filter using a vacuum
manifold and a vacuum pump. Filters were then washed
with 7 mL of filter-sterilized 210 millimolar (mM)
KH,PO,, buffer (pH 6.0) followed by 3 mL of 25 mM
KH,PO4 buffer (pH 7.2). Filter cups were then sealed on
the bottom, after adding 2.5 mL of BHI growth medium.
After mixing. 60 jjJL aliquots were taken from each filter
cup and transferred to a 96-well PCR plate (aliquots
taken at time 0; TO). The cups were sealed on Hie top and
incubated for 16 hr at 37 °C and 230 rpm. Another set
of 60 |jJL aliquots were taken after 16 hr of incubation
(aliquots taken after 16 hr of incubation; T16). For each
set of aliquots. the PCR plate was sealed, centrifuged
and placed in a My Cycler™ thermal cycler for 20 min at
95 °C for heat lysis. For samples with high levels of dirt
(wipes with Arizona test dust), a 1:10 dilution in PCR-
grade water was performed prior to running PCR.
When the RV-PCR protocol was performed manually, all
liquid handling was effectuated with serological pipettes
or micro-pipettes. In the semi-automated version of the
-------
protocol, with the exception of the initial sample spiking,
the Perkin-Elmer Janus robotic platform performed all the
liquid handling steps required to implement the RV-PCR
method including mixing and transferring buffer from
sample extracts to filtration media for spore collection,
washes on the filters, and adding growth medium to the
filter cups for culturing and sampling cultures for PCR
analysis.
Sample in conical
tube with support
Add extraction
buffer, vortex
Rapid Viability
PCR
1
Collect sample by
filtration in filter
cup
Add media to
filter-cup
Vortex filter cups,
TO aliquot for
PCR
IncubateShrs
Vortexfiltercups,
T9 aliquot for
PCR
DNAcleanup,
Promega kit
q-PCRonTOand
T9aliquots,data
reporting
Traditional
Viability
Total
processing
time for 24
samples is
<15hr
Figure 1. Summary of the optimized RV-PCR protocol steps and pictures of the equipment used to process
samples.
-------
Optimized Rapid-Viability PCR
In the optimized protocol, the RV-PCR method was
implemented according to the protocol described
above, with the exception of the introduction of a 10
min vortexing step of the filter cups prior to aliquoting
samples for TO and T-endpoint (at the end of the
incubation period). The incubation time was reduced
from 16 hr to 9 hr (T9) and sample aliquots were
manually processed using the Promega magnetic bead-
based DNA extraction and purification kit (MagneSil®
Blood Genomic, Max Yield System; Promega, Catalog
number MD1360).6Detailed protocols are provided in
Appendix A and B. Briefly, 1 mL of each sample was
transferred from the filter cup into a 2 mL eppendorf
tube, followed by addition of 600 |jL of bead mix
(combined lysis buffer and bead mix) and 360 |jL
additional lysis buffer. Sample, buffer and bead mix
were mixed by pipetting and tubes were mounted
on a tube rack interfacing with a magnet (see Figure
2). Beads with attached DNA were attracted to the
magnet and the supernatant was removed by pipetting.
An additional lysis with 360 |jL of lysis buffer was
conducted with mixing by pipetting and removal of the
supernatant. Two washes with 360 |jL of salt solution
were then performed, followed by mixing and removal
of the supernatant. Finally, two washes with 360 |jL of
alcohol wash solution were performed with mixing and
supernatant removal. Beads were allowed to air-dry for
2 min, followed by transfer of the tube rack from the
magnetic support to a hot plate and heating at 80°C until
samples/beads are dry (between 15 and 45 min). DNA
elution/concentration was then performed by adding
200 |j,L of elution buffer while sample tubes remained
on the hot plate. The sample with buffer was mixed and
transferred to the magnetic support, and the supernatant
with eluted DNA was recovered (typically 80 |jL). A ten-
fold dilution of the eluted sample in PCR-grade water
was systematically performed prior to running PCR.
The DNA extraction and purification procedure was
performed according to the manufacturer's protocol,6
with the exception of the first step in which the sample
is mixed with bead mix. Since the sample volume
used in the RV-PCR method is larger (1 mL) than the
sample volume used in the manufacturer's technical
bulletin (200 uL), after receiving guidance from the
manufacturer, the volume of beads was increased from
130 to 600 uL.
A summary of liquid transfers is provided in Figure 3.
Figure 2. Magnetic rack used to process samples with
the Promega DNA extraction and purification kit.
B, anthracis DNA Standards for
Real-time PCR
DNA standards were generated for the Ba Ames strain.
DNA was extracted from cultured cells using a complete
a MasterPure™ Complete DNA and RNA Purification
Kit (Epicentre® Biotechnologies Inc.) and DNA
concentration was measured with a Qubit™ fluorimeter
using the PicoGreen™ assay (Invitrogen™, Quant-iT™
dsDNA HS assay kit for Qubit fluorimeter, Part Number
Q32854). Standard concentrations ranging from 1 ng/
|jL to 1 fg/|j.L were prepared in PCR-grade water. Seven
10-fold dilutions, ranging from 5 ng per 25-(oL PCR to 5
fg per 25-(oL PCR, were run with each set of PCR plates.
The ROX reference dye contained in the ABI universal
mastermix was used to normalize the fluorescent reporter
signal.
Real-time PCR
Five microliter sample aliquots were transferred to a
96-well PCR plate with 20 (oL of PCR mix. PCR mix
was prepared for each primer-probe set according to
conditions detailed in Tables 10-11 and in the PCR
protocol in Appendix F, using TaqMan™ 2X Universal
Master Mix (ABI cat. 4305719). After mixing and
centrifugation, PCR was run using the ABI 7500 Fast
platform (Applied Biosystems 7500 Fast Real-Time
PCR System). Cycle parameters were as follows: 2 min
at 50°C forUracil-N-glycosilase (UNO) incubation, 10
min at 95°C for AmpliTaq™ gold activation, followed
by 45 amplification cycles (5 s at 95°C for denaturation
and 20 s at 60°C for annealing/extension). For the assay
optimization study, each PCR was run in triplicate. For
RV-PCR, each sample was analyzed against each of the
3 primer/probe sets (one PCR was run for each sample
against each primer/probe set, 3 replicate samples were
analyzed for each set of experimental conditions).
TO control samples
In the initial part of the work (RV-PCR protocol using
a 16 hr incubation step), a 60 |jL aliquot was taken out
of each filter cup at TO, heat-lysed for 20 min at 95 °C
-------
and analyzed by PCR with the Ba assays to provide TO
background Ct values (these have consistently been
>45).
After optimization of the RV-PCR protocol with the
addition of the magnetic bead-based DNA extraction
and purification, TO aliquots were handled differently,
to match the processing performed at T9 and to also
equate the aliquots withdrawn at TO and T9 (see section
4.3.4, Development of a TO Control Protocol). In this
procedure, three 120 |jL aliquots are taken out of filter
cups at TO, mixed with 900 (oL of BHI and spiked with
500 pg of extracted Bg DNA. Each of the 120 (oL aliquot
is pipetted out of the filter cup and 100 |jL is dispensed
into a 2 mL eppendorf tube, to follow BSL3 pipetting
guidelines. The TO samples chosen are 3 replicates
spiked at the highest spiking concentration: 102 or 103
spore level. The three 1 mL samples are then processed
following the magnetic bead-based DNA extraction
and purification method described above. The extracted
samples are then analyzed using the Ba assays in order
to determine whether any background is present; a
Bg assay is used as a positive control. Ct values at TO
have consistently been >45 for all Ba assays in control
experiments.
RV-PCR results interpretation
For the RV-PCR method, the endpoint PCR Ct of < 36
with a ACt (Ct[TO]-Ct[T-endpoint]) > 9 were set as cut-
off values for a positive detection of viable Ba spores.
It should be noted that most laboratories, including the
CDC's Laboratory Response Network (LRN), use the
PCR Ct value of <40 as a cut-off value for a positive
detection of Ba spores (live or heat-killed). We set a
more stringent Ct cut-off value to test the reliability and
robustness of the RV-PCR method. Depending upon
the end user requirement and the phase of response
during an event, a lower ACt (Ct[TO]-Ct[T-endpoint])
> 6 (to represent at least a two log difference in DNA
concentration), and a higher end point (PCR Ct of ^39)
could be set.
Sample spiking, plating
1
Conical tube: Wipe + 100 ul of spiking suspension + 20 ml buffer
j
Vortexing
1
13 ml transferred to filter cup, filtered through filter
i
Filter washes
i
1
2.5 ml of BH! growth medium is added to filter cup
120 |iL are aliquotted out of filter cup for TO plate
Incubation
1 ml aliquotted out of filter cup for processing with Promega kit
SQu.L is eluted after DNA clean up
10 nL of the eluted sample is used and mixed with 90 ul of water (10-fold dilution}
1
15 uL of the dilution is used for PCR (3 assays x 5 u,L reaction)
Figure 3. Summary of liquid handling steps performed during the optimized RV-PCR protocol.
-------
Final RV-PCR for
Final manual and semi-automated RV-PCR protocols
were designed for the analysis of real-world samples.
The detailed protocols are located in Appendices A and
D.
-------
4.0
Results and Discussion
4.1 Assay Development
4.1.1. In silico Analysis
Signatures for Ba real-time PCR were ranked and
selected in silico using the following parameters:
signature specificity against all available sequences
in GenBank®(U.S. National Institutes of Health's
genetic sequence database), virulence gene association,
availability of prior assay screening data, and amplicon
characteristics. Signatures from multiple sources were
considered, including the Department of Homeland
Security (DHS) Public Safety Actionable Assays
Program (PSAA), the Homeland Security Advanced
Research Project Agency (HSARPA) Program, and
EPA's NHSRC. Published assays, signatures developed
at LLNL for other projects, as well as new signatures
developed for this project were also evaluated (Table 2).
Erosion/Non-verification analysis provides a prediction
of potential cross-reactions and misses as signatures
are compared against all available genomic sequence
data. The LLNL KPATH microbial database contains
all available full-length microbial genomes and is
updated weekly. The LLNL erosion analysis software
uses algorithms that compare signatures as primer
pairs or triplets against all sequence in the large
KPATH database and identifies targets with sufficient
percent match to each oligomer to represent a potential
hybridization. Erosion results include predicted false
positives against non-target sequences, and non-
verifications which are predicted false negatives against
target sequences. True positives are also identified.
Table 2. Summary of All Bacillus anthracis
Signatures Analyzed in Silico
Source
HSARPA
DHS / PSAA
Publication7
RNA Viability project (LLNL-generated)
EPA (Provided by Dr. Sanjiv Shah)
EPA (LLNL-generated)
Total
Ba
Signatures
18
0
1
17
2
6
44
The output of this analysis was a computational
prediction of virulent Ba strain detection for 44 candidate
assays ranked for predicted selectivity, amplicon size,
and gene target. These files are too large to include here
in a Microsoft® Office Word document but a summary of
signatures selected for this project is presented in Table
3. Ten assays (3 for the chromosome, 4 for the pXOl
plasmid and 3 for the pXO2 plasmid) were selected for
optimization based on the in silico analysis.
Table 3. List of 10 Bacillus anthracis Assays Selected
After in Silico Analysis
Signature
Name
BC1
BC2
BC3
BP11
BP12
BP13
EPA-1
BP21
BP22
EPA-2
Target
Chromosome
Chromosome
Chromosome
pXOl
plasmid
pXOl
plasmid
pXOl
plasmid
pXOl
plasmid
pXO2
plasmid
pX02
plasmid
pX02
plasmid
Signature
ID
1776135
1776147
1776201
1776125
2186847
1776115
2212179
2148341
2148343
2212180
Gene Target
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
membrane protein,
putative
capsule biosynthesis
protein cap b
4.7.2. Assay Optimization
A dedicated ABI7500 Fast thermocycler was purchased
from Applied BioSystems, Inc. (ABI) with NHSRC
funds for this RV-PCR project. PCR for both assay
development and Rapid Viability method was always
performed using this platform. Selected signatures were
ordered from Biosearch Technologies, Inc. (Novato,
CA) (all primers and probes were HPLC-purified and
probes were modified as 5'-6FAM and 3'-BHQl). Ba
Ames genomic standards were prepared and initially
characterized using both Nanodrop™ UV spectrometry
and Qubit® nuorimetry with the PicoGreen® double-
stranded DNA quantification assay. Initial tests showed
-------
that the concentration of double-stranded Ba Ames
DNA measured using the PicoGreen assay was typically
25% of the concentration measured using the Ultra
Violet (UV) spectrophotometric method and Nanodrop
spectrometer (see Table 4), which was expected since the
PicoGreen assay only detects double-stranded DNA. All
DNA concentrations were therefore measured using the
PicoGreen assay for the entire study.
Table 4. Measurement of Bacillus anthracis Ames
DNA Concentration by Nanodrop™ UV Spectrometer
and Qubit® Fluorimeter based methods
Table 6. Initial Screening of Bacillus anthracis pXOl
Plasmid Assays BP11, BP12, BP13 and EPA-1
Method
Nanodrop UV
Spectrometer
Qubit
Fluorimeter,
PicoGreen
Assay
DNA (ng/uL)
Replicate 1 Replicate 2 Replicate 3 Average
3.80
1.24
3.90
1.11
3.90
1.16
3.87
1.17
An initial experiment was conducted to test each primer/
probe set with a series of eight 10-fold dilutions of Ba
Ames DNA in triplicate, following PCR conditions
provided by NHSRC (see Materials and Methods and
PCR conditions in Appendix F). This initial experiment
showed that the primer/probe sets that had been
optimized with this PCR mix and platform (EPA-1 and
EPA-2) performed according to specifications, but that
other primer/probe sets would need optimization (see
Tables 5-7).
Table 5. Initial Screening of Bacillus anthracis
Chromosome Assays BC1, BC2 and BC3
DNA(pg)
5000
500
50
5
0.5
0.05
0.005
Average Ct*
BC1
29.1
32.7
NDT
NDT
NDT
NDT
NDT
BC2
27.9
32.4
42.9
NDT
NDT
NDT
NDT
BC3
(selected)
23.3
27.0
39.8
41.9
NDT
NDT
NDT
DNA(pg)
5000
500
50
5
0.5
0.05
0.005
Average Ct*
BP11
29.7
33.5
NOT
NOT
NOT
NOT
NOT
BP12
28.7
33.0
32.6
NOT
NOT
NOT
NOT
BP13
33.8
36.3
39.7
NOT
NOT
NOT
NOT
EPA-1
(selected)
20.7
23.1
26.4
29.9
33.4
37.2
NOT
* Average Ct (n=3)
Acronyms: Ct, cycle threshold; n, number of replicates; NDT, no signal
detected
Table 7. Initial Screening of Bacillus anthracis pXO2
Plasmid Assays BP21, BP22, and EPA-2
DNA(pg)
5000
500
50
5
0.5
0.05
0.005
Average Ct*
BP21
27.7
39.1
40.6
42.3
44.1
NDT
NDT
BP22
24.7
28.3
34.8
41.4
43.7
44.4
NDT
EPA-2
(selected)
18.5
21.4
24.7
28.4
31.9
35.4
NDT
* Average Ct (n=3)
Acronyms: Ct, cycle threshold; n, number of replicates; NDT, no signal
detected
Based on this initial sensitivity screening, EPA-1 and
EPA-2 assays were immediately selected for the pXOl
and pXO2 plasmid targets respectively. The BC3 assay
was the best performer in the chromosomal assay
category in the initial screening and it was therefore
selected for optimization. Sequence and amplicon size
information for the selected assays are provided in Table
* Average Ct (n=3)
Acronyms: Ct, cycle threshold; n, number of replicates; NDT, no signal
detected
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Table 8. Nucleotide Sequences and Amplicon Size for Selected Bacillus anthracis RV-PCR Assays
Assay
(alias)
Chromosome
(BC3)
pXOl phi sin id
(EPA-1)
pXO2 plasmid
(EPA-2)
LLNL
Signature
Number
1776201
2212179
2212180
Forward Primer
TTTCGATGATT
TGCAATGCC
GCGGATAGCG
GCGGTTA
TGCGCGAATGA
TATATTGGTTT
Reverse Primer
TCCAAGTTACAG
TGTCGGCATATT
TCGGTTCGTTAA
ATCCAAATGC
GCTCACCGATAT
TAGGACCTTCTTTA
Probe
ACATCAAGTCAT
GGCGTGACTACCCAGACTT
ACGACTAAACCG
GATATGACATTA
AAAGAAGCCCTTAA
TGACGAGGAG
CAACCGATTAAGCGC
Amplicon
Length (bp)
105
101
77
Acronyms: bp, base pair units; LLNL, Lawrence Livermore National Laboratory
Primer and probe titrations were performed on the BC3
assay in triplicate, using 5 ng and 500 pg of extracted
Ba Ames DNA template. The probe concentration was
varied between 0.08 and 0.20 uM, while the primer
concentration was varied between 0.4 and 1.0 uM per
reaction. The conditions chosen were 1.0 uM of primer
and 0.08 uM of probe per reaction, since they provided
good sensitivity and matched the conditions used for the
EPA-1 assay (see highlight in Table 9).
The final PCR conditions for the selected assays are
described in detail in Tables 10-12 below.
Reagents:
• Primers and probes
• TaqMan® 2X Universal Master Mix with UNO and
AmpliTaq® Gold DNA polymerase (ABI catalogue
Number 4305719)
• Molecular Biology grade distilled water, RNase- and
DNase-free (Teknova catalogue Number W3350)
Table 9. Optimization of Primer and Probe
Concentration for the BC3 Assay
Table 10. PCR Mix for pXO2 (EPA-2)
Primer/Probe Set
Primer (uM)
0.4
0.4
0.4
0.6
0.6
0.6
0.8
0.8
0.8
1.0
1.0
1.0
Probe (uM)
0.08
0.12
0.20
0.08
0.12
0.20
0.08
0.12
0.20
0.08
0.12
0.20
Average Ct*
5 ng DNA
20.4
20.6
20.7
19.8
19.7
20.0
19.7
19.7
19.9
19.5
19.6
19.6
500 pg DNA
23.1
23.2
23.2
22.8
22.7
23.0
22.8
22.6
22.5
22.6
22.7
22.5
* Average Ct (n=3)
Selected PCR conditions are highlighted in blue.
Acronyms: Ct, cycle threshold; n, number of replicate reactions
Reagent
TaqMan 2X Universal Master Mix
Forward primer, 25 uM
Reverse primer, 25 uM
Probe, 2 uM
Molecular Biology Grade Water
Template DNA
TOTAL
Volume
GiL)
12.5
0.3
0.3
1
5.9
5
25
Final
Concentration
(uM)
IX
0.3
0.3
0.08
N/A
Variable
-------
Table 11. PCR Mix for Chromosome (BC3) and
pXOl (EPA-1) Primer/Probe Sets
Reagent
TaqMan 2X Universal Master Mix
Forward primer, 25 uM
Reverse primer, 25 uM
Probe, 2 uM
Molecular Biology Grade Water
Template DNA
TOTAL
Volume
(uL)
12.5
1
1
1
4.5
5
25
Final
Concentration
(uM)
IX
1.0
1.0
0.08
N/A
Variable
Equipment:
• ABI7500 fast thermocycler
• Optical fast 96-well plates (ABI, cat. Number
4366932)
• Optical adhesive plate covers (ABI, cat. number
4311971)
Table 12. PCR Thermal Cycling Conditions for All 3 Primer/Probe Sets
Steps
Temper-
ature
Time
UNG incubation
Hold*
50°C
2 min
AmpliTaq Gold
activation
Hold*
95°C
10 min
PCR , 45 cycles
Denatur-ation*
95°C
5 s
Annealing/extension *
60°C
20s
*Fast Ramp: 3.5oC/s up and 3.5oC/s down
The performance of the three selected assays is
summarized in Table 13 and Figure 4 below. After
optimization, all three assays enabled detection of less
than 10 copies (50 fg) of extracted Ba Ames DNA. The
genome copies are based on an estimated genome size of
5.5 Mb. The chromosomal assay provided slightly higher
Ct values than the plasmid assays, which is an expected
result since more plasmid copies are typically produced.
Table 13. Performance of Optimized BC3, EPA-
1, and EPA-2 PCR Assays Tested With Bacillus
anthracis Ames DNA
DNA(pg)
5000
500
50
5
0.5
0.05
Genome
Copies
829000
82900
8290
829
82.9
8.29
Average Ct*
BC3
19.4
22.6
26.5
30.6
34.6
38.5
EPA-1
20.7
23.1
26.4
29.9
33.4
37.2
EPA-2
18.5
21.4
24.7
28.4
31.9
35.4
* Average Ct (n = 3)
Acronyms: Ct, cycle threshold; n, number of replicate reactions
-------
V(Cfuanwenw)> •) Ml X • J1.M1; M' • O.MM
rti
II
c
.voo
0,92 1,9? -?^J 3,92 491 S 92
LogJC of Genome Copies
Figure 4. PCR efficiency of selected chromosomal and plasmid Bacillus anthrads Ames assays.
Additional experiments were then performed with the
assays in order to insure their suitability for Rapid-
Viability PCR. Inhibition by the BHI growth medium
was tested by comparing the PCR performance of
the Ba Ames DNA dilutions in PCR-grade water and
BHI medium. Results showed that variations in cycle
threshold values induced by the presence of growth
medium in the PCR were generally below 1 Ct (see
Table 14).
Table 14. Change in average Ct value for Bacillus
anthrads Ames Assays with BHI Medium in the PCR,
Compared to Water
DNA (pg)
5000
500
50
5
0.5
0.05
Change in Average Ct*
EPA-1**
-0.6
1.1
0.2
0.8
0.5
1.0
EPA-2**
0.7
0.2
0.7
0.7
0.5
-1.3
BC3**
0.5
-0.1
-0.9
-0.5
-0.5
3.7
* Average Ct (n=3)
** Average Ct (BHI) - Average Ct (water)
Acronyms: pg, picogram
The effect of cell debris on the PCR was also evaluated
by comparing Ba Ames DNA dilutions in PCR-grade
water and in a lysed culture of Bg (109 cells/mL). Results
showed that variations in cycle threshold values induced
by the presence of cell debris in the PCR were generally
below 2 Cts (see Table 15).
Table 15. Change in average Ct value forBadllus
anthrads Ames Assays with lysed culture of Bacillus
atrophaeus (109 cells/mL) in the PCR, Compared to
Water
DNA(pg)
5000
500
50
5
0.5
0.05
Change in Average Ct*
EPA-1**
-1.1
-0.7
-1.2
-1.5
-1.6
-1.0
EPA-2**
0.4
2.9
2.4
2.1
2.0
3.1
BC3**
1.8
0.1
-1.4
-0.1
1.3
1.8
* Average Ct (n=3)
* * Average Ct (Bg cell lysate) - Average Ct (water)
Acronyms: pg, picogram
4.7.3. Selectivity Study
Concentrations of extracted DNA templates were
measured using the PicoGreen assay, and diluted to
appropriate stock concentrations using PCR-grade
water. PCR plate lay-outs were prepared to run triplicate
reactions for each assay against each target and near-
neighbor DNA (two concentrations were run for each
template: 500 fg per reaction and 50 fg per reaction
for targets and 5 pg and 500 fg per reaction for near
neighbors). Three positive controls (extracted Ba Ames
DNA) and three negative controls (PCR-grade water)
were run for each assay on each PCR plate. Results
summarized in Tables 16 and 17 showed that all assays
detected Ba targets, as expected, and that no cross
-------
reactivity was observed with near neighbors, with the
exception of Bacillus cereus 03BB102 for both plasmid
assays and Bacillus cereus 03BB108 for the pXO2 assay.
These interactions of the plasmid assays with Bacillus
cereus strains were predicted by the in-silico analysis.
Table 16. PCR Assay Selectivity Study Using a Panel of Bacillus anthracis Strain DNA
Targets
B. anthracis
B. anthracis
B. anthracis
B. anthracis
B. anthracis
B. anthracis
B. anthracis
B. anthracis
B. anthracis
B. anthracis
B. anthracis
B. anthracis
B. anthracis
Strain
Turkey 32
A0149
A0248
V770-NP-1R
BalOlS
SK-102
Bal035
K3
Ames
PAK-1
RA3
Vollum IB
Sterne
Average Ct* with indicated PCR assays and DNA concentrations
BC3
50 fg
36.5 (0.7)
37.2 (0.6)
36.6 (0.7)
36.4(0.4)
38.1 (1.5)
37.1(0.7)
37.4(1.2)
37.1 (0.3)
35.9(0.5)
36.8(1.7)
38.4(0.5)
38.8(0.4)
36.7(0.6)
500 fg
32.7(0.2)
32.7(0.5)
32.9 (0.3)
33.0(0.3)
33.7(0.2)
32.9 (0.2)
33.3 (0.5)
33.4(0.2)
33.0(0.3)
33.0(0.2)
33.9(0.3)
34.3 (0.3)
33.1 (0.1)
EPA-1
50 fg
34.7 (0.4)
35.6(0.3)
35.6(0.8)
35.1(0.8)
36.2(1.0)
35.7(0.2)
35.0(0.1)
35.5(0.5)
35.1(0.1)
34.8(0.2)
35.6(0.3)
37.1 (0.9)
35.0(0.2)
500 fg
31.3(0.1)
31.9(0)
31.7(0.2)
31.2(0.1)
32.5 (0.2)
32.2 (0.2)
31.4(0.1)
31.7(0.1)
31.4(0.1)
31.7(0.1)
32.5(0.1)
33.3(0.1)
31.5(0.3)
EPA-2
50 fg
34.0 (0.3)
35.8(0.5)
35.9(1.0)
NOT (pX02-)
35.5(0.5)
35.0(0.2)
34.6 (0.7)
35.9(1.5)
35.1(0.9)
34.0 (0.2)
35.5 (0.8)
35.0(0.2)
NOT
(pX02-)
500 fg
30.2(0.1)
31.9(0.3)
31.5(0.2)
NOT (pX02-)
32.6 (0.2)
30.7 (0.4)
30.3(0.1)
30.7(0.2)
31.4(0.3)
30.6 (0.2)
31.9(0.3)
31.5(0.3)
NOT
(pX02-)
* Average Ct (SD, n = 3)
Acronyms: Ct, cycle threshold; fg, femtogram; n, number of replicates; NDT, no signal detected; PCR, polymerase chain reaction; SD, standard
deviation
Table 17. PCR Assay Selectivity Study Using a Panel of Near-Neighbor Bacillus species DNA
Near
Neighbors
B. cereus
B. cereus
B. thuringiensis
B. thuringiensis
B. thuringiensis
B. cereus
B. cereus
B. thuringiensis
B. thuringiensis Al Hakam
B. cereus
B. cereus
B. cereus
B. thuringiensis israelensis
B. thuringiensis kurstaki
B. thuringiensis morrisoni
Strain
S2-8
3A
HD1011
97-27
HD682
E33L
D17
HD571
FM1
03BB102
03BB108
Average Ct* with indicated PCR assays and DNA concentrations
BC3
500 fg
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
5pg
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
EPA-1
500 fg
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
32.4(0.3)
NDT
NDT
NDT
NDT
5pg
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
29.0 (0)
NDT
NDT
NDT
NDT
EPA-2
500 fg
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
35.0(0.2)
37.6 (0.6)
NDT
NDT
NDT
5pg
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
NDT
31.8(0.3)
35.2 (0.7)
NDT
NDT
NDT
* Average Ct (SD, n = 3)
Acronyms: Ct, cycle threshold;
deviation
fg, femtogram; n, number of replicates; NDT, no signal detected; PCR, polymerase chain reaction; SD, standard
-------
4.2. Select Agent Laboratory Set Up
The CDC Select Agent Program regulates the
possession, use, and transfer of biological agents and
toxins that could pose a severe threat to public health
and safety (select agents). While select agent operations
were already approved and ongoing at LLNL, a new
laboratory dedicated to the EPA work was set up, and a
team with RV-PCR experience was assembled. Any work
activity involving select agents requires a permit from
CDC which is specific to people, locations, organisms
and protocols. Internal LLNL approvals for select agent
work were also requested and granted.
Team members applied for and received clearances from
the Department of Justice. Training included select agent
handling, shipping, receiving, and transferring, as well as
a Select Agent Human Reliability Program certification
(yearly psychological and medical evaluations, as well
as drug testing). All personnel also received the anthrax
vaccine.
Dedicated equipment was purchased with NHSRC funds
and installed in the dedicated Select Agent laboratory,
providing both manual and semi-automated RV-PCR
capabilities for Select Agents (see pictures in Figure
5). Instrumentation purchased includes an Applied
Biosystems, Inc. ABI7500 Fast PCR platform, a
Beckmann-Coulter (Beckmann-Coulter, Inc., Brea, CA)
centrifuge with safety cups, a New-Brunswick shaker
incubator (New Brunswick Scientific, Edison, NJ), a
VWR refrigerator, a Perkin-Elmer Janus robotic platform
for semi-automated liquid handling, and a custom
HEPA-filtered enclosure for the Janus built by E-N-G
Mobile Systems Inc. The Class II BioSafety Cabinet
(BSC), freezer, and standing incubators were provided
by LLNL. Both BSC and robot enclosures are certified
semi-annually as a best management practice.
4.3. Rapid Viability-Polymerase
Chain Reaction (RV-PCR) Method
Development
The major test variables for RV-PCR protocol
development included inoculum density (103 to 101
spores per sample), presence/absence of reference
background debris, presence/absence of heat-killed target
spores, and presence/absence of non-target organisms
(including Bg and Pa). Ba spores were spiked directly
onto/into wipe, air filter, and water samples rather than
collected from surfaces. The determination/evaluation
of the efficiency of spore removal from sample matrices
was outside the scope of this study.
RV-PCR protocols were evaluated for the following
criteria: accuracy with plate counts from spiking
solutions, selectivity, sensitivity (< 50 spores per
sample), throughput capacity (24-48 samples per day
with manual and semi-automated methods), turn-around
time for results (< 24 hr), and absence of PCR and/or
growth inhibition (a 1:10 dilution of the samples was
used as needed to overcome inhibition).
RV-PCR experiments were performed according to
the Materials and Methods section and to the detailed
protocols provided in Appendices A and B. Experiments
consisted of a minimum of three replicates per treatment
and routinely used 4 to 6 replicates per treatment. Each
replicate sample was analyzed once against each PCR
assay (BC3, pXOl, and pXO2). Initial and final cycle
thresholds (Ct0 and Ctf) from RV-PCR assays were
used to determine whether viable spores were present
in the sample. Since Ct0 values were consistently >
45, RV-PCR results during the protocol evaluation and
optimization phase were expressed in term of average
Ctf value and corresponding standard deviation for
each set of conditions (sample type, spore level, added
Figure 5. Pictures of the EPA-dedicated Select Agent laboratory at LLNL. On the left is the manual capability
(Class II BioSafety Cabinet), and on the right is the automated capability (Janus robotic platform and custom,
HEPA-filtered enclosure from E-N-G Mobile Systems Inc.).
-------
background or debris). For the verification phase,
Ct0, Ctp and ACt were reported for each sample-type
analyzed. Traditional viability analysis was used to
quantify the level of spores spiked onto/into sample
materials in each experiment. Two dilutions were plated
and 3 replicate plate counts were used to calculate
the standard deviation of the spore level applied, as
described in the Materials and Methods.
4.3.7. Development of Manual RV-PCR
Spore levels of 1000, 100 and 10 CPU were spiked on
clean wipes in triplicate. Table 18 summarizes the Ct
values (average of 3 replicate samples) obtained at the
endpoint (T16) for the 3 Ba assays at each spore level.
The Ct values at TO were >45 cycles and Ct values at
T16 were <35 for all samples, confirming a detection
limit at the 10 spore level for clean wipes with the
manual method. Spore levels were determined by
plating.
Table 18. Manual RV-PCR at T16 on Clean Wipes
Spiked With 3 Spore Levels
CFU/Sample
22
110
1100
Average Ct*
EPA-1
33.0 (2.0)
31.1(2.0)
29.9(1.8)
EPA-2
26.3 (1.7)
25.3(1.0)
25.3 (0.8)
BC3
33.4(3.3)
33.0(2.5)
31.0(2.5)
*Average Ct (SD, n = 3)
Acronyms: CPU, colony forming units; Ct, cycle threshold; n, number
of replicates; SD, standard deviation
Ultra-fine Arizona test dust was then added to clean
wipes at a level of 250 mg per wipe. Spore levels of 100
and 10 CPU were spiked and 8 replicate samples were
used for each spore level. Initial PCR results at T16 from
experiments performed without diluting the aliquots
were 'non-detect' for dirty wipes (spiked with the AZ
Test Dust). Ten fold dilutions of all dirty wipes sample
aliquots (T16 aliquots) were then prepared in PCR-
grade water and positive PCR detection was achieved.
PCR inhibition from the Arizona Test Dust is believed
to mainly come from metal oxides. Analysis of the
chemical composition performed by the manufacturer
indicates that the material consists of: SiO2 (68 to 76%),
A12O3 (10 to 15%), Fe2O3 (2 to 5%), Na2O (2 to 4%),
CaO (2 to 5%), MgO (1 to 2%), TiO2(0.5 to 1.0%), and
K2O (2 to 5%). The fine particles present in this dust
also enhance the reactivity of this material by creating
a large specific area. Table 19 summarizes the Ct values
(average of 8 replicates) obtained at the endpoint (T16)
for the 3 Ba assays at each spore level.
The Ct values at TO were >45 cycles and Ct values at
T16 were <35 for all samples, confirming a detection
limit at the 10 spore level for dirty wipes with the
manual method.
Table 19. Manual RV-PCR at T16 on Clean and Dirty
Wipes Spiked With 2 Spore Levels
CFU/Sample
12
110
Wipe
Clean
Dirty
Clean
Dirty
Average Ct*
EPA-1
29.9 (2.6)
34.8(2.1)
22.6 (0.6)
27.9 (4.0)
EPA-2
24.8(2.9)
32.9(1.6)
22.6(1.1)
23.7(1.2)
BC3
27.1(0.6)
33.3(1.7)
24.0 (0.4)
29.1(2.6)
*Average Ct (SD, n = 3)
*For dirty wipes, a 1:10 dilution of the heat-lysed samples was
performed in PCR-grade water prior to running PCR to reduce
inhibition from the AZ test dust. Ct values were not corrected from the
dilution factor.
Acronyms: CPU, colony forming units; t, cycle threshold; n, number of
replicates; SD, standard deviation
Spore levels of 1000, 100 and 10 CPU were spiked on
clean filters in triplicate. Table 20 summarizes the Ct
values (average of 3 replicate samples) obtained at the
endpoint (T16) for the 3 Ba assays at each spore level.
The Ct values at TO were >45 cycles and Ct values at
T16 were <35 for all samples, confirming a detection
limit at the 10 spore level for clean air filters with the
manual method.
Table 20. Manual RV-PCR at T16 on Clean Filters
Spiked With 3 Spore Levels
CFU/
Sample
20
120
1000
Average Ct*
EPA-1
28.3(1.9)
24.8(1.2)
23.1 (0.5)
EPA-2
22.2 (4.0)
21.0(1.5)
20.9 (0.3)
BC3
27.8(0.5)
28.2(0.3)
25.2(0.3)
*Average Ct (SD, n = 3)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of
replicates; CFU, colony forming units
Spore levels of 1000, 100 and 10 CFU were spiked on
dirty filters. Dirty filters used in this experiment were
collected from a Portable Sampling Unit (PSU) in
-------
Phoenix, AZ in November 2008. Table 21 summarizes
the Ct values (average of 8 replicate samples) obtained
at the endpoint (T16) for the 3 Ba assays at each spore
level.
The Ct values at TO were >45 cycles and Ct values at
T16 were <35 for all samples, confirming a detection
limit at the 10 spore level for dirty air filters with the
manual method.
Table 21. Manual RV-PCR at T16 on Dirty Air Filters
Spiked With 3 Spore Levels
CFU/Sample
8
116
1100
Average Ct*
EPA-1
32.8(2.5)
31.7(3.5)
30.6(1.8)
EPA-2
30.4(4.9)
26.0 (5.4)
22.6(1.6)
BC3
33.6 (0.9)
29.5(2.1)
31.8(1.7)
*Average Ct (SD, n = 8)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of
replicates; CFU, colony forming units
Spore levels of 1000, 100 and 10 CFU were spiked in
20 mL clean laboratory water samples (deionized, sterile
water) and dirty water samples (10 mg/L humic acid
and 10 mg/L ferrous sulfate spiked in deionized, sterile
water) in triplicate. Table 22 summarizes the Ct values
(average of 3 replicate samples) obtained at the endpoint
(T16) for the 3 Ba assays at each spore level.
Wipes, 10 spore level
PSU Filters, 10 spore level
EPA1
EPA2
BC3
The Ct values at TO were >45 cycles and Ct values at
T16 were <35 for all samples, confirming a detection
limit at the 10 spore level for both clean and dirty water
with the manual method.
Figure 6 summarizes the data presented in this section
for both clean and dirty sample types, with an inoculum
level of 10 Ba CPU/sample. Detection limits at the 10
CPU/sample level were demonstrated on both clean
and dirty samples (all Ct values were below 35). A 1:10
dilution of the sample was required prior to running PCR
on dirty wipes in order to reduce inhibition from the AZ
test dust. Other samples did not require any dilution.
Table 22. Manual RV-PCR at T16 on Clean and Dirty
Water Spiked With 3 Spore Levels
CFU/
Sample
78
780
7800
Water
Clean
Dirty
Clean
Dirty
Clean
Dirty
Average Ct*
EPA-1
27.4(1.5)
28.6 (2.0)
26.6 (2.7)
27.7(1.3)
29.5 (3.3)
28.7(1.3)
EPA-2
24.1 (1.1)
22.1(3.1)
22.6 (2.2)
22.0(1.3)
23.9 (2.4)
24.2(1.0)
BC3
29.1 (2.0)
28.6(1.4)
30.3 (0.2)
28.3 (1.4)
30.0(0.8)
29.4(3.3)
*Average Ct (SD, n = 3)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of
replicates; CFU, colony forming units
Water, 10 spore level
10mg/L
ferrous
sulfate
and
humic
acid
EPA1
EPA2
BC3
• All Ct values at T = 0 are >45
• All Ct values at T = 16 hrs are <35
• Ct values are averages of 3 replicates for
clean samples and 6 replicates for dirty
samples.
• Addition of debris on wipes and air filters
Increased Ct values (typically 3-7 Cts), mainly
due to PCR inhibition.
• Addition of ferrous sulfate and humic acid in
water(10 mg/L) did not significantlyaffectCt
values.
Figure 6. Summary of Ct values obtained with the manual RV-PCR protocol on both clean and dirty sample
types (wipes, water and air filters) using a spiking level of 10 Bacillus anthrads spores per sample.
-------
Manual RV-PCR experiments to evaluate the influence
of high levels of live non-target background on the limit
of detection for Ba were started with clean wipes. Spore
levels of 100 and 1000 spores per sample were spiked
on clean wipes in the presence of live Pa only (106 CPU/
sample), as well as in the presence of live Bg (103 CPU/
sample) combined with live Pa (106 CPU/sample). Table
23 summarizes the Ct values (average of 5 replicate
samples) obtained at the endpoint (T16) for the 3 Ba
assays at each spore level (the Ct at time zero was >45
cycles for all samples).
All Ct values obtained in the presence of live Pa were
<36. All Ct values obtained in the presence of both live
Pa and Bg were <39 (in all cases, 2 out of 3 assays had
Ct values <35), confirming the robustness of the RV-
PCR approach in the presence of high levels of non-
target organisms (Ct values above 36.0 are highlighted
in blue in Table 23). Note that the EPA-2 assay allowed
detection of the 100 live Ba Ames spores level in a
combined background of up to 106 live Pa and 105
live Bg (Ct[100 spores, T16]=35.0, Ct [650 spores,
T16]=33.4).
Table 23. Manual RV-PCR at T16 on Clean Wipes
Spiked With 2 Spore Levels in the Presence of B.
globigii and P. aeruginosa Background
Ba
CFU/
Sample
100
650
Non-Target
Organisms
106/sample
Pa
106/sample
Pa&
103/sample
Bg
106/sample
Pa
106/sample
Pa&
103/sample
Bg
Average Ct*
EPA-1
35.8(3.4)
38.3 (5.6)
35.2(2.1)
37.2 (4.4)
EPA-2
23.9(1.7)
25.4(5.1)
26.8(5.8)
23.4(2.3)
BC3
34.5(1.2)
33.3 (3.5)
30.8 (2.0)
33.0(3.8)
*Average Ct (SD, n = 5)
Ct values above 36.0 are highlighted in blue.
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of
replicates; CFU, colony forming units
Spore levels of 10 and 100 CFU were spiked on clean
filters in the presence of live Pa only (106 CPU/sample),
as well as in the presence of live Bg (103 CPU/sample)
combined with live Pa (106 CPU/sample). Table 24
summarizes the Ct values (average of 5 replicate
samples) obtained at the endpoint (T16) for the 3 Ba
assays at each spore level (the Ct at time zero was >45
cycles for all samples).
All Ct values obtained in the presence of live Pa were
<35. All Ct values obtained in the presence of both live
Pa and Bg were <41 (in all cases, 2 out of 3 assays had
Ct values <35), confirming the robustness of the RV-
PCR approach in the presence of high levels of non-
target organisms (Ct values above 36.0 are highlighted
in blue in Table 24). Note that the EPA-2 assay allowed
detection of the 100 live Ba Ames spores level in a
combined background of up to 106 live Pa and 105
live Bg (Ct[85 spores, T16]=39.0, Ct [330 spores,
T16]=34.7).
Table 24. Manual RV-PCR at T16 on Clean Filters
Spiked With 2 Spore Levels in the Presence of B.
globigii and P. aeruginosa Background
Ba CFU/
Sample
85
330
Non-Target
Organisms
106/sample
Pa
106/sample
Pa&
KP/sample
Bg
106/sample
Pa
106/sample
Pa&
KP/sample
Bg
Average Ct*
EPA-1
33.4(0.2)
33.5(5.0)
31.4(1.7)
33.8(3.3)
EPA-2
30.9(2.5)
31.3(5.3)
24.4(1.3)
29.7(1.0)
BC3
33.6 (2.0)
40.1 (4.2)
32.3(1.1)
38.1(3.1)
*Average Ct (SD, n = 5)
Ct values above 36.0 are highlighted in blue.
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of
replicates; CFU, colony forming units
Spore inocula at the 100 spore level were spiked in clean
laboratory water (filtered and sterilized) in the presence
of live Pa only (106 CPU/sample), as well as in the
presence of live Bg (103 CPU/sample) combined with
live Pa (106 CPU/sample). Table 25 summarizes the Ct
values (average of 5 replicate samples) obtained at the
endpoint (T16) for the 3 Ba assays at each spore level
(the Ct at time zero was >45 cycles for all samples).
All Ct values obtained in the presence of live Pa were
<35. All Ct values obtained in the presence of both live
Pa and Bg were <41 (in all cases, 2 out of 3 assays had
Ct values <35), confirming the robustness of the RV-PCR
approach in the presence of high levels of non-target
organisms (Ct values above 36.0 are highlighted in blue
in Table 25).
-------
Table 25. Manual RV-PCR at T16 on Clean Water
Spiked With 2 Spore Levels in the Presence of B.
globigii and P. aeruginosa Background
Ba CFU/
Sample
123
250
Non-Target
Organisms
lO'/sample
Pa
lO'/sample
Pa&
lOVsample
Bg
106/sample
Pa
106/sample
Pa&
lOVsample
Bg
Average Ct*
EPA-1
27.2 (5.2)
39.0(1.9)
29.6 (2.5)
33.2(1.5)
EPA-2
20.6(1.0)
29.7(1.9)
22.2(1.4)
29.6 (3.0)
BC3
26.6 (0.2)
40.1(3.0)
27.9(1.9)
39.9 (4.2)
Table 26. Manual RV-PCR at T16 on Clean Wipe,
Air Filter and Water Samples Spiked With 2 Live
Spore Levels in the Presence of Heat-Killed Bacillus
anthracis Ames Spores
*Average Ct (SD, n = 5)
Ct values above 36.0 are highlighted in blue.
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of
replicates; CPU, colony forming units
In order to investigate the effect of high levels of dead
Ba Ames on the detection of low levels of the same
live organism, an experiment was performed in which
two levels of live Ba spores were spiked on clean
wipes, air filters and water samples in the presence of
106 heat-killed Ba Ames spores/sample. A stock of Ba
Ames spores (106 CFU/mL confirmed by plate counts)
was killed by autoclaving three times at 126 °C for 30
min. Six 100 |aL aliquots were then plated on solid BHI
medium and no colonies were observed after 48 hr,
confirming that all spores were dead. A manual RV-PCR
experiment was performed, in which each sample type
(wipes, filters, and water) was spiked with 106 heat-
killed Ba spores and 75 and 400 live Ba spores. Four
replicates were generated for each sample type and spore
level. Table 26 summarizes the Ct values (average of 4
replicate samples) obtained at the endpoint (T16) for the
3 Ba assays at each spore level.
All Ct values obtained in the presence of heat-killed
Ba spores were <37 (in all cases, 2 out of 3 assays had
Ct values <35), confirming the detection of low levels
of live spores in a high background of dead spores
(decontamination scenario) (Ct values above 36.0 are
highlighted in blue in Table 26).
Background Ct values (average of 4 replicate samples)
for 106 heat-killed spores/sample only (no live spores)
after 16 hr of growth were: 42.89, 44.02, and 40.57 for
the BC3, EPA-1, and EPA-2 assays respectively. Such
background may originate from DNA leaking from dead
cells.
Sample
Type
Wipes
Air
Filters
Water
CFU/
Sample
75
400
75
400
75
400
Average Ct*
EPA-1
33.3 (7.2)
29.2(0.5)
37.0 (7.7)
31.3(6.8)
29.2 (4.9)
29.3 (5.8)
EPA-2
20.2(0.8)
20.5(1.5)
19.6(1.2)
20.1 (2.4)
20.6(0.8)
19.7(1.6)
BC3
27.8(1.4)
25.5(1.0)
26.2 (2.9)
27.0 (2.3)
27.0 (2.2)
24.3(2.1)
* Average Ct (SD, n = 4)
Ct values above 36.0 are highlighted in blue.
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of
replicates; CFU, colony forming units
4.3.2 Development of Semi-automated RV-PCR
Since no Select Agent work had previously been
performed with robotics in our facility, a work
observation by the Biosafety Officer and Select Agent
Manager was required to authorize the new robotics
protocol at LLNL. The observation was recorded in
the safety documents and approval to operate the Janus
robotic platform with Select Agents was granted. Figure
7 shows the Janus platform and the HEPA-filtered
enclosure in the Select Agent laboratory.
The Janus robotic platform was programmed to perform
all the liquid handling steps required to implement
the RV-PCR method, with the exception of sample
spiking. Semi-automated protocols include mixing and
transferring buffer from sample extracts to filtration
media for spore collection, as well as performing washes
on the filters, adding growth medium to filter cups for
culturing and sampling cultures for PCR analysis.
-------
Figure 7. Janus robotic platform and custom HEPA-
filtered enclosure in the Select Agent laboratory.
Spore levels of 100 and 10 CFU were spiked on all
sample types in triplicate. Table 27 summarizes the Ct
values (average of 3 replicate samples) obtained at the
endpoint (T16) for the 3 Ba assays.
The Ct values at TO were >45 and Ct values at T16 were
<35 for all samples, confirming a detection limit at the
10 spore level for clean sample types with the semi-
automated method.
The same experiment was then performed with dirty
samples. Dirty wipes were generated by adding 250
mg of ultra-fine AZ test dust to clean wipes, dirty air
filters were collected from a subway location (Boston,
Massachusetts, 02/2008) and dirty water was generated
by spiking clean and sterile laboratory water with humic
acid and ferrous sulfate at a level of 10 mg/L. Table
28 summarizes the Ct values (average of 5 replicate
samples) obtained at the endpoint (T16) for the 3 Ba
assays (Ct values above 36.0 are highlighted in blue).
The Ct values at TO were >45 and Ct values at T16 were
<35 for dirty filter and dirty water samples, confirming
a detection limit at the 10 spore level for these sample
types in the presence of dirt.
PCR inhibition was observed in the presence of AZ test
dust on the wipe samples, similar to what was observed
with the manual protocol. Systematic 1:10 dilutions of
the dirty wipe samples were therefore prepared and re-
analyzed by PCR. With 250 spores per sample, 2 out of
3 assays had Ct values <35. With 36 spores per sample,
Ct values were between 36.3 and 37.3 for all 3 assays.
Additional experiments were then conducted in order
to optimize the protocol (see the Method Optimization
section). It was found that the up and down pipetting
performed by the Janus platform did not provide enough
mixing of the cultured filter cups prior to aliquoting the
T16 samples. Vortexing filter cups for 10 min prior to
aliquoting the T16 samples generated lower Ct values.
Table 27. Semi-automated RV-PCR at T16 on Clean
Wipe, Air Filter and Water Samples Spiked With 2
Spore Levels
Sample
Type
Wipes
Air Filters
Water
CFU/
Sample
36
244
36
244
36
244
Average Ct*
EPA-1
32.0(1.8)
20.1(0.6)
29.2(1.7)
22.2(3.8)
26.9 (0.7)
21.8(0.4)
EPA-2
21.9(1.2)
19.3(2.1)
20.7(1.7)
19.7(2.5)
20.4 (0.4)
21.4(0.3)
BC3
24.8(1.1)
26.3 (1.4)
26.6(0.1)
24.0(1.2)
25.0(0.8)
23.8 (0.7)
*Average Ct (SD, n = 3)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of
replicates; CFU, colony forming units
-------
Table 28. Semi-automated RV-PCR at T16 on Dirty
Wipe, Air Filter and Water Samples Spiked With 2
Spore Levels
Sample
Type
Wipes**
Air
Filters
Water
CFU/
Sample
36
244
36
244
36
244
Average Ct*
EPA-1
37.3 (3.2)
38.0(5.4)
28.4(2.2)
27.6 (3.6)
28.0(3.1)
29.2 (4.5)
EPA-2
36.3(4.1)
31.1(5.3)
21.4(0.9)
20.3(1.1)
20.8(1.6)
21.5(1.5)
BC3
36.4(3.6)
28.4(8.2)
23.3(1.3)
26.2(3.5)
25.4(2.3)
25.6(1.1)
*AverageCt(SD, n = 5)
**A 1:10 dilution of the sample was made prior to running PCR in
order to reduce inhibition from the AZ test dust. Ct values were not
corrected from this dilution factor.
Ct values above 36.0 are highlighted in blue.
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of
replicates; CFU, colony forming units
Table 29. Semi-automated RV-PCR at T16 on Clean
Wipe, Air Filter and Water Samples Spiked With 2
Spore Levels in the Presence of Heat-Killed Bacillus
anthrads Spore Background
Sample
Type
Wipes
Air
Filters
Water
CFU/
Sample
45
151
45
151
45
151
Average Ct*
EPA-1
31.8(1.1)
27.3 (0.3)
30.5(1.7)
27.3 (0.3)
29.2 (0.6)
27.1 (1.2)
EPA-2
31.1(1.5)
26.3 (0.3)
29.8(1.4)
27.0 (0.9)
29.0(1.8)
26.3 (0.6)
BC3
32.7(1.0)
29.0 (0.6)
31.1(1.0)
29.9(1.0)
31.2(2.1)
29.3 (1.0)
*AverageCt(SD, n = 4)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of
replicates; CFU, colony forming units
In order to investigate the effect of high levels of dead
Ba Ames on the detection of low levels of the same
live organism, we performed an experiment in which
two levels of live Ba spores were spiked on clean
wipes, air filters and water samples in the presence of
106 heat-killed Ba Ames spores/sample. A stock of Ba
Ames spores (106 CFU/mL confirmed by plate counts)
was killed by autoclaving three times at 126 °C for 30
min. Six 100 |j,L aliquots were then plated on solid BHI
medium and no colonies were observed after 48 hr,
confirming that all spores were dead. A semi-automated
RV-PCR experiment was performed, in which each
sample type (wipes, filters, and water samples) was
spiked with 106 heat-killed Ba spores and 45 and 151
live Ba spores. Four replicate samples were generated for
each sample type and spore level. Table 29 summarizes
the Ct values (average of 4 replicate samples) obtained
at the endpoint (T16) for the 3 Ba assays at each spore
level.
All Ct values obtained in the presence of heat-killed
Ba spores were <35, confirming the robustness of the
RV-PCR method in the presence of high dead target
organisms (decontamination scenario). Baseline Ct
values (average of 4 replicate samples) at TO for 106
heat-killed spores/sample only were: 45, 45, and 41.7 for
the BC3, EPA-1, and EPA-2 assays, respectively.
Semi-automated RV-PCR in the presence of high levels
of live non-target organisms was then conducted. Spore
levels of 10 and 100 CFU were spiked on clean samples
in the presence of live Pa only (106 CPU/sample), as
well as in the presence of live Bg (103 CPU/sample)
combined with live Pa (106 CPU/sample). Tables 30-32
summarize the Ct values (average of 4 replicate samples)
obtained at the endpoint (T16) for the 3 Ba assays at
each spore level.
The Ct values at TO were >45 for all samples. Ct values
obtained in the presence of live Pa were <35. Ct values
obtained in the presence of combined Pa and Bg were
also <35, confirming the robustness of the RV-PCR
method in the presence of high levels of live non-target
organisms.
-------
Table 30. Semi-automated RV-PCR at T16 on Clean
Wipes Spiked With 2 Spore Levels in the Presence of
B. globigii and P. aeruginosa Background
Ba
CFU/
Sample
36
274
Non-
Target
Organisms
106/sample
Pa
106/sample
Pa&
lOVsample
Bg
106/sample
Pa
106/sample
Pa&
103/sample
Bg
Average Ct*
EPA-1
21.0(0.3)
23.1(0.3)
20.7(1.2)
21.0(0.4)
EPA-2
20.0 (0.4)
21.4(0.2)
20.0 (0.5)
19.3 (0.4)
BC3
24.1 (2.7)
25.1(0.6)
24.4(0.5)
23.1(0.7)
* Average Ct (SD, n = 4)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of
replicates; CFU, colony forming units
Table 32. Semi-automated RV-PCR at T16 on Clean
Water Spiked With 2 Spore Levels in the Presence of
B. globigii and P. aeruginosa Background
Ba
CFU/
Sample
36
274
Non-Target
Organisms
106/sample
Pa
106/sample
Pa&
lOVsample
Bg
lO'/sample
Pa
106/sample
Pa&
103/sample
Bg
Average Ct*
EPA-1
21.9(1.2)
23.4(0.8)
19.8(0.7)
19.9(0.1)
EPA-2
20.7(0.8)
22.0 (0.6)
19.1 (1.1)
18.8(0.5)
BC3
24.5(1.2)
25.6(0.9)
26.9 (6.7)
23.0 (0.2)
*Average Ct (SD, n = 4)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of
replicates; CFU, colony forming units
Table 31. Semi-automated RV-PCR at T16 on Clean
Filters with 2 Live Spore Levels in the Presence of B.
globigii and P. aeruginosa Background
Ba
CFU/
Sample
36
274
Non-Target
Organisms
106/sample
Pa
106/sample
Pa&
lOVsample
Bg
lO'/sample
Pa
106/sample
Pa&
lOVsample
Bg
Average Ct*
EPA-1
20.9(1.4)
22.7(0.8)
20.2(1.1)
20.9 (0.4)
EPA-2
19.7(1.2)
21.4(0.4)
19.3(1.2)
19.2(0.3)
BC3
23.6 (2.0)
25.3 (0.9)
23.2(1.3)
23.2 (0.4)
* Average Ct (SD, n = 4)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of
replicates; CFU, colony forming units
Both manual and semi-automated RV-PCR methods
were showed to have detection limits at the 10 spore
level. Figure 8 provides a direct comparison of Ct
values obtained on clean samples using manual and
semi-automated RV-PCR methods. The semi-automated
protocol was found to provide slightly lower Ct values
(up to 4 Cts) than the manual protocol, most probably
due to improved pipetting precision and reproducibility.
The detection limit was maintained for both manual and
semi-automated methods in the presence of challenges
including dirt, high levels of heat-killed Ba Ames
spores, and high levels of live, combined non-target
Bacillus species and vegetative cells (although Ct values
increased in the presence of challenges, they remained
<35 in most cases). As will be seen in the next section,
modifications of the protocols were implemented in
order to further improve Ct values. While the semi-
automated protocol performed well on clean samples, it
was found that for dirty wipes, additional mixing of the
cultured samples in filter cups by vortexing was required
after the incubation step in order to obtain optimal
results.
-------
40
35
30
25
0 20
15
10
5
0
40 T
35 \
30
25
Clean Wipes, 10 spore level
EPA1
EPA2
BC3
Clean PSU Filters, 10 spore level
EPA1
EPA2
BC3
40
35
Clean Water, 10 spore level
EP.A1
EPA2
BC3
1 All Ct values at T = 0 are >45
• All Ct values at T = 16 hrs are <35
• Automated protocol typically
provided lower Ct values (typically 0-4
Cts)and smaller error (factor 2 on
standard deviation).
Figure 8. Summary of Ct values obtained with manual and semi-automated RV-PCR protocols on clean samples
(wipes, filters and water) spiked at the 10 Ba spore level.
4.3.3. Method Optimization
4.3.3.1. Improvement of Sample Mixing
after Incubation
After noticing high Ct values in the presence of debris
with the semi-automated protocol, vortexing of the
filter cups at T16 was evaluated to improve sample
mixing before aliquoting after overnight growth. Results
obtained without any vortexing were compared to results
obtained after implementing 10 min of vortexing of the
filter cups prior to aliquoting the sample after incubation.
The data, summarized in Table 33, showed a reduction
of both Ct values (2-4 Ct values) and standard deviations
for detection using 36 spores when the vortexing step
was implemented (see Figure 9).
Table 33. Semi-automated RV-PCR at T16 on Clean
Wipe, Air Filter and Water Samples Spiked with 36
Bacillus anthracis Ames Spores
Sample
Type
Wipes
Air
Filters
Water
Protocol
Vortexing
No
Vortexing
Vortexing
No
Vortexing
Vortexing
No
Vortexing
Average Ct*
EPA-1
32.0(1.8)
35.3 (4.6)
29.2(1.7)
33.9(2.8)
26.9 (0.8)
30.8(0.9)
EPA-2
21.9(1.2)
23.2(0.5)
20.7(1.7)
23.1 (0.4)
20.4 (0.4)
22.2(1.4)
BC3
24.8(1.1)
31.5(1.7)
26.6(0.1)
30.9 (0.4)
25.0(0.8)
28.0(1.6)
*Average Ct (SD, n = 3)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of
replicates
-------
45
40
Clean wipes, 10 spore level
T
EPA1
EPA2
45
40
Clean PSu filters, 10 spore level
45
40
Clean water, 10 spore level
EPA1
EPA2
•Vortexing filter cups for 10 min prior
to aliquotting the T = 16 hrs samples
reduced Ct values by up to 5 Cts.
EPA1
EPA2
BC3
Figure 9. Summary of Ct values obtained with the semi-automated RV-PCR protocol on clean samples with and
without vortexing filter cups prior to aliquoting sample for PCR. Samples were spiked at the 10 spore level.
4.3.3.2. Shortening of the RV-PCR Method Endpoint
for More Rapid Detection of Bacillus anthracis Spores
A manual RV-PCR experiment was conducted with clean
wipe, filter and water samples spiked at the 10 and 100
Ba spore levels in order to assess whether live Ba spores
could be detected after 9 hr of incubation (four replicate
samples were processed for each set of conditions). In
this experiment, the only changes made in the protocol
were the reduction of the incubation time from 16 to 9
hrs, and the addition of 10 min of vortexing of the filter
cups prior to aliquoting the samples.
Results obtained after heat lysis at T9 are summarized
in Table 34 below (no dilution of the samples was
performed) (Ct values above 36.0 are highlighted in
blue). At a level of 10 CPU/sample, the BC3 and EPA-1
assays did not provide any measurable Ct values and
the EPA-2 assay provided Ct values above 34. At a level
of 100 CPU/sample, the BC3 assay did not typically
generate measurable Ct values and the EPA-1 and EPA-2
assays provided Ct values typically above 30, with high
standard deviations. It was therefore determined that an
additional DNA extraction step was required in order to
generate reliable Ct values for low spore levels at T9.
Table 34. Manual RV-PCR at T9 on Heat-Lysed
Extracts of Clean Wipe, Air Filter and Water
Samples Spiked With 2 Spore Levels
Sample
Type
Wipes
Air
Filters
Water
CFU/
Sample**
28
272
28
272
28
272
Average Ct*
EPA-1
NDT
36.7 (2.4)
NDT
37.1 (3.6)
NDT
38.6(3.0)
EPA-2
NDT
30.9(2.1)
34.8(3.1)
31.4(3.5)
34.3 (3.4)
33.7(3.3)
BC3
NDT
NDT
NDT
NDT
NDT
39.6 (4.2)
*Average Ct (SD, n = 4)
"Samples were lysed with heat lysis and analyzed with PCR (no
dilution was performed)
Ct values above 36.0 are highlighted in blue.
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of
replicates; CFU, colony forming units; NDT, no Ct detected
-------
Two manual RV-PCR experiments (one performed on
clean wipes, filters and water, and the other performed on
dirty wipes, filters and water) were then performed (the
method is described in the Materials and Method section
and a detailed protocol is provided in Appendix A) in
order to demonstrate the potential of the introduction
of a DNA extraction and purification step to shorten
the method endpoint without compromising the limit
of detection. Two spore levels were tested (10 CPU/
sample and 100 CPU/sample) and 4 replicate samples
were processed for each set of conditions. Dirty wipes
were generated by adding 250 mg of ultra-fine AZ test
dust to clean wipes dirty air filters were collected from a
subway location (Boston, Massachusetts, 02/2008) and
dirty water was generated by spiking clean and sterile
laboratory water with humic acid and ferrous sulfate at a
level of 10 mg/L.
In these experiments, the manual RV-PCR method
was used according to the regular protocol, with the
exception of the introduction of a 10 min vortexing step
of the filter cups prior to aliquoting samples for TO and
T-endpoint. The incubation time was reduced from 16
hr to 9 hr and sample aliquots were manually processed
using the Promega DNA extraction and purification
kit. Since the protocol starts from a 1 mL sample and
the final elution is performed using a 200 |j,L volume, a
5-fold concentration of the DNA is achieved. In addition,
a cleaner DNA sample is expected for PCR analysis
since growth medium and debris are removed.
Tables 35 and 36 summarize the results obtained with
clean and dirty sample types respectively. Both data
sets showed that levels down to 10 spores per sample
were detected after 9 hr of incubation using the new
protocol, even in the presence of debris. The total
manual processing time from start to finish for 24
samples was <15 hr (25 min vortexing, 1.5 hr for set up
of filter cups, 9 hr of incubation, 2 hr for DNA extraction
and purification, and 1.5 hr for PCR). A 1:10 dilution of
the DNA extracted and purified from the samples was
necessary to obtain consistent Ct values in the presence
of ultra-fine Arizona test dust, but all Ct values were <35
without any correction from the dilution factor.
Table 35. Manual RV-PCR at T9 on Clean Wipe,
Air Filter and Water Samples Spiked With 2 Spore
Levels and Processed with the DNA Extraction and
Purification Protocol
Sample
Type
Wipes
Air
Filters
Water
CFU/
Sample**
40
130
40
130
40
130
Average Ct*
EPA-1
22.7(0.5)
20.1(1.1)
24.0 (0.7)
21.7(1.2)
23.7(0.6)
23.2(1.9)
EPA-2
20.6 (0.3)
20.5 (0.2)
21.5 (0.7)
19.7(0.7)
21.5(0.6)
21.3 (2.0)
BC3
23.7(0.1)
21.2(1.0)
24.7 (0.9)
22.7(0.8)
24.3 (0.5)
24.2(1.9)
* Average Ct (SD, n = 4)
* * Samples were processed using the DNA extraction and purification
protocol
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of
replicates; CFU, colony forming units
Table 36. Manual RV-PCR at T9 on Dirty Wipe,
Air Filter and Water Samples Spiked With 2 Spore
Levels and Processed with the DNA Extraction and
Purification Protocol
Sample
Type
Wipes***
Air
Filters
Water
CFU/
Sample**
40
130
40
130
40
130
Average Ct*
EPA-1
33.8(2.3)
27.6 (0.8)
24.1(1.3)
21.4(2.6)
23.9(1.5)
22.2(1.9)
EPA-2
31.4(1.5)
25.4(0.5)
22.2(1.7)
20.1(1.4)
21.5(1.2)
20.3 (1.7)
BC3
35.1 (2.9)
29.4(0.8)
25.5(1.8)
23.0(1.4)
24.7(1.4)
23.1(1.6)
* Average Ct (SD, n = 4)
* * Samples were processed using the DNA extraction and purification
protocol.
***A 1:10 dilution of the sample was prepared prior to running PCR
in order to reduce inhibition from the AZ test dust. Ct values were not
corrected from this dilution factor.
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of
replicates; CFU, colony forming units
-------
An additional experiment with wipe samples was
performed to assess whether the incubation time could
be further reduced to 8 hr and whether the improved
RV-PCR method would work in all the verification
conditions (clean samples, dirty samples, clean samples
with heat-killed Ba background, and clean samples with
live non-target background). Wipe samples were spiked
at the 10 live Ba Ames spore level and six replicate
samples were analyzed for each set of conditions.
Samples were processed using the semi-automated
RV-PCR protocol with 10 min of vortexing of the filter
cups prior to aliquoting samples. The incubation time
was reduced to 8 hr and samples were cleaned using
the Promega DNA extraction and purification protocol.
At TO, a 60 uL aliquot was taken out of each filter cup
in order to perform a heat lysis control. At T8, a 60 uL
aliquot was taken out of each filter cup to perform heat
lysis, and a 1 mL aliquot was taken for DNA extraction
and purification.
As shown in Table 37, heat lysis at TO did not generate
any measurable Ct values, confirming the absence of Ba
DNA background. Various protocols were compared for
the analysis of the T8 samples. PCR was performed after
heat lysis, after heat lysis and a 1:10 dilution, after DNA
extraction and purification, and after DNA extraction
and purification followed by 1:10 dilution. Results for
all 4 analysis methods are presented in Table 38. Heat
lysis did not generate Ct values below 37 after 8 hr of
incubation. The addition of a 1:10 dilution often led to
measurable Ct values, but these were typically in the
high 30s. Processing of the samples using the Promega
DNA extraction and purification kit did not consistently
lead to measurable Ct values at T8, while the addition
of a 1:10 dilution after the Promega extraction and
purification did. This experiment showed the necessity
to perform the Promega DNA extraction and purification
protocol to generate reliable data with the shortened
incubation time and the necessity to perform a 1:10
dilution to reduce PCR inhibition coming from dirt and
debris, chemical residues from wipe samples, and/or
residual chemicals from the magnetic bead-based DNA
extraction and purification protocol, including alcohol.
The data summarized in Table 38 also showed that the
10 spore level could be detected in all cases but that
Ct values were generally closer to 35 (35.7 for the
chromosomal assay in the presence of AZ test dust, after
1:10 dilution). In light of these results, it was decided
to use a 9 hr incubation time as the end point for the
single laboratory method verification. A flow chart of the
optimized protocol used for the verification phase of the
project is provided in Figure 10.
Table 37. Manual RV-PCR After Heat Lysis at TO on
Wipes Spiked with 28 Bacillus anthracis Spores
Clean
Dirty (250 mg
AZ test dust)
lO'/sample
heat-killed
Ba Ames
lO'/sample
Pa&
lOVsample
Bg
Average Ct*
EPA-1
NOT
NOT
NOT
NOT
EPA-2
NOT
NOT
NOT
NOT
BC3
NOT
NOT
NOT
NOT
*Average Ct (SD, n = 6)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of
replicates; CPU, colony forming units; NDT, no Ct detected
Table 38. Manual RV-PCR at T8 on Wipes Spiked With 28 Bacillus anthracis Spores
T8 PCR condition
Heat Lysis
Heat Lysis + 1:10 Dilution
Promega kit
Promega kit +1:10 dilution
Heat Lysis
Heat Lysis + 1:10 Dilution
Promega kit
Promega kit + 1:10 dilution
Heat Lysis
Heat Lysis + 1:10 Dilution
Promega kit
Promega kit +1:10 dilution
Assay
EPA1
EPA2
BC3
Average Ct With:*
Clean Sample
NDT
34.8(1.3)
NDT
33.1(0.8)
40.0 (3.6)
32.8(1.2)
NDT
31.9(1.4)
NDT
37.9(1.5)
NDT
33.1(1.9)
250mgAZ
test dust
NDT
38.6(1.8)
NDT
33.9(2.2)
NDT
37.3 (1.4)
NDT
31.8(2.2)
NDT
43.3 (2.0)
NDT
35.7(3.3)
lO'/sample heat-
killed Ba Ames
NDT
34.1(0.8)
35.6(3.9)
31.3(1.4)
37.3(1.0)
32.3 (0.4)
28.4(1.8)
29.8(1.4)
NDT
36.5(1.0)
35.1(1.9)
32.9 (2.2)
lO'/sample Pa &
lOVsample Bg
41.5(1.5)
33.8(0.6)
36.0(2.3)
31.1(1.7)
37.8(3.0)
31.5(1.4)
28.9 (2.4)
29.1 (1.8)
43.1 (0.4)
35.7(1.2)
36.0(2.8)
32.3 (2.0)
* Average Ct (SD, n = 6)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CFU, colony forming units; NDT, no Ct detected
-------
Sample in conical
tube with support
Add extraction
buffer, vortex
Rapid Viability PCR
Collect sample by
filtration in filter
cup
Add media to filter-
cup
Vortex filter cups,
TO aliquot for PCR
Incubate 9 hrs
Vortex filter cups,
T9 aliquot for PCR
Heat lysis
_L
DNA clean up,
Promegakit
q-PCR on TO and T9
aliquots, data
reporting
Traditional Viability
•Total
processing
time for 24
samples is 14
15 hrs
• 3-4 hrs of
processing
time added for
each set of 24
samples
Figure 10. Optimized RV-PCR method overview. Items in red indicate optimization steps.
4.3.4. Development of a TO Control Protocol
After adding the new DNA extraction and purification
step to the RV-PCR protocol, a new control had to
be implemented at TO. A series of experiments were
performed in order to: 1) assess whether any detectable
Ba DNA was present at TO when analyzing TO samples
with the new DNA extraction and purification protocol,
and 2) determine the most effective way to implement
a TO control, the challenge being that a 1 mL aliquot
cannot be withdrawn from the filter cup at TO when
the total volume of growth medium is 2.5 mL without
significantly affecting the spore growth and the limit of
detection at the endpoint.
The first TO control experiment consisted in processing
1 mL TO aliquots using the Promega DNA extraction and
purification protocol. The goal was to determine if any
detectable Ba DNA was present at TO. Clean wipes were
spiked with increasing levels of Ba Ames spores ranging
from 10 CPU/sample up to 106 CPU/sample. A set of
clean wipes was also spiked at the 106 heat-killed Ba
Ames spore level and another set was spiked at the 100
live Ba Ames spore level in a background of 106 heat-
killed Ba Ames spores. Three replicates were processed
for each set of experimental conditions using the RV-
PCR protocol. One hundred microliters of samples
spiked at the 103 spore level were plated at TO (plate
counts in 100 uL were: 41, 28 and 26 colonies) and T9
(plate counts in 100 uL were too numerous to count on
all 3 plates) as a control. Sixty microliter aliquots were
removed out of each filter cup in order to perform heat
lysis at TO. One milliliter aliquots were then removed
out of each filter cup at TO and processed by heat lysis
or using the Promega DNA extraction and purification
protocol (undiluted and 10-fold diluted samples were
analyzed by PCR using all 3 Ba assays).
-------
Neither the heat lysis nor the DNA extraction and
purification method (undiluted or diluted) generated any
measurable Ct values at TO. The only exceptions were
a few high Ct values (1 out of 3 replicates), bolded in
Tables 39 and 40, which correspond to a spiking level
of 106 live spores per sample. No TO Ct values were
measured on samples spiked with 106 heat-killed Ba
Ames spores.
These results confirm that neither heat lysis nor the
Promega DNA extraction and purification protocol lyse
Ba spores at TO and that background at TO does not
affect experiments conducted with spiking levels below
105 CPU/sample. It should be noted that no background
was measured on the samples spiked with heat-killed
spores, which indicates that spore killing by autoclaving
did not induce significant DNA leakage from the Ba
spores.
Finally, these results confirm the cleanliness of the spore
preparation used in this study. For samples spiked at the
106 spore level, each 5 uL PCR would contain ~ 6500
spores, assuming 100% recovery. At this level, heat lysis
did not provide any measurable Ct, and DNA extraction
and purification provided Ct values around 40.
Table 39. Manual RV-PCR at TO on Wipes Processed with DNA Extraction and Purification of on 1 mL Aliquots
CFU/Sample
10 live BaAines
102 live Ba Ames
103 live Ba Ames
104 live Ba Ames
10s live Ba Ames
10' live Ba Ames
10' heat-killed Ba Ames
102 live Ba Ames +
10' heat-killed Ba Ames
Average Ct*
BC3
NOT, NOT, NOT
NOT, NOT, NOT
NOT, NOT, NOT
NOT, NOT, NOT
NOT, NOT, NOT
NOT, NOT, NOT
NOT, NOT, NOT
NOT, NOT, NOT
EPA-1
NOT, NOT, NOT
NOT, NOT, NOT
NOT, NOT, NOT
NOT, NOT, NOT
NOT, NOT, NOT
NOT, NOT, 42.8
NOT, NOT, NOT
NOT, NOT, NOT
EPA-2
NOT, NOT, NOT
NOT, NOT, NOT
NOT, NOT, NOT
NOT, NOT, NOT
NOT, NOT, 43.2
NOT, NOT, 38.0
NOT, NOT, NOT
NOT, NOT, NOT
* Average Ct (SD, n = 3)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units; NDT, no Ct detected
Table 40. Manual RV-PCR on Wipes Processed with DNA Extraction and Purification on 1 mL Aliquot at TO
Followed by 1:10 Dilution of the Extracted Samples
CFU/Sample
10 live Ba Ames
102 live Ba Ames
103 live Ba Ames
104 live Ba Ames
10s live Ba Ames
10' live Ba Ames
10' heat-killed Ba Ames
102 live Ba Ames +
10' heat-killed Ba Ames
Average Ct*
BC3
NDT, NDT, NDT
NDT, NDT, NDT
NDT, NDT, NDT
NDT, NDT, NDT
NDT, NDT, NDT
NDT, NDT, 43.6
NDT, NDT, NDT
NDT, NDT, NDT
EPA-1
NDT, NDT, NDT
NDT, NDT, NDT
NDT, NDT, NDT
NDT, NDT, NDT
NDT, NDT, NDT
NDT, NDT, 38.9
NDT, NDT, NDT
NDT, NDT, NDT
EPA-2
NDT, NDT, NDT
NDT, NDT, NDT
NDT, NDT, NDT
NDT, NDT, NDT
NDT, NDT, NDT
NDT, NDT, 42.4
NDT, NDT, NDT
NDT, NDT, NDT
*Average Ct (SD, n = 3)
Acronyms: Ct, cycle threshold; SD,
standard deviation; n, number of replicates; CPU, colony forming units; NDT, no Ct detected
-------
A second control experiment was performed in which the
goal was to equate sample volumes and concentrations
at TO and T9 (see Table 41 for details). Clean wipes
were spiked with increasing levels of Ba Ames spores
ranging from 10 CPU/sample up to 106 CPU/sample. A
set of clean wipes was also spiked with 106 heat-killed
Ba Ames spores and another set was spiked with 100
live Ba Ames spores in a background of 106 heat-killed
Ba Ames spores. Three replicates were processed for
each set of experimental conditions using the RV-PCR
protocol. One hundred microliters of samples spiked
at the 103 spore level were plated from filter cups at TO
(plate counts in 100 uL were 53, 52 and 33 colonies) and
T9 (plate counts in 100 uL were too numerous to count
on all 3 plates) as a control. Sixty-microliter aliquots
were removed out of each filter cup at both TO and T9 in
order to perform heat lysis. Another 60 uL aliquot was
removed out of each filter cup at TO in order to perform
a modified version of the Promega DNA extraction and
purification protocol in which 60 uL of the sample are
mixed with 940 uL of TE buffer prior to processing.
One-milliliter aliquots were also removed out of each
filter cup at T9 and analyzed using the Promega DNA
extraction and purification protocol. Bg carrier DNA was
added in the samples processed with the Promega DNA
extraction and purification protocol in order to provide a
positive control. The presence of Bg DNA was evaluated
with a Bg assay available at LLNL.
Table 41. Experiment Design for Aliquot Equivalency between TO and T9 Samples
TO
60 uL sample + 940 uL TE
(Total sample volume is
ImL)
DNA Extraction and
Purification
No dilution
PCR
(Results in Table 42)
TO, carrier DNA
concentration
(Bg)
30 pg/mL
150pg/mL(5X
concentration by DNA
extraction and purification)
150pg/mL
0.750 pg / rxn (5 uL sample)
(Results in Table 42)
T9
1 in 1 sample
DNA extraction and
purification
1:17 dilution
PCR
(Results in Table 45)
T9 carrier DNA
concentration
(Bg)
500 pg/mL
2500 pg/mL (5X
concentration by DNA
extraction and purification
150pg/mL(l:17 dilution)
0.750 pg / rxn (5 uL sample)
(Results in Table 45)
Acronyms: uL, microliter; pg, picogram; rxn, reaction
Heat lysis analysis at TO did not provide any measurable
Ct values for any assay on any sample.
DNA extraction and purification with the Promega
protocol at TO did not provide any measurable Ct values
for any assay on any sample (Table 42).
Table 42. Manual RV-PCR on Wipes Processed with DNA Extraction and Purification at TO
(Table 41 Column 1)
CFU/Sample
10 live Ba Ames
102live BaAmes
103 live BaAmes
104 live BaAmes
10s live Ba Ames
10' live BaAmes
10' heat-killed Ba Ames
1 02 live BaAmes +
10' heat-killed Ba Ames
Average Ct*
BC3
NOT
NOT
NOT
NOT
NOT
NOT
NOT
NOT
EPA-1
NOT
NOT
NOT
NOT
NOT
NOT
NOT
NOT
EPA-2
NOT
NOT
NOT
NOT
NOT
NOT
NOT
NOT
Bg
NOT
NOT
NOT
NOT
NOT
37.9 (2.0)
37.3 (0.6)
36.9 (0.4)
*Average Ct (SD, n = 3)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CFU, colony forming units; NDT, no Ct detected
-------
Heat lysis analysis at T9 did not allow the detection of
the 10 Ba spore level but generated measurable Ct values
for spore levels above 100 CPU/sample (Table 43).
Table 43. Manual RV-PCR at T9 on Wipes with Heat Lysis
CFU/Sample
10 live BaAmes
102 live Ba Ames
103 live Ba Ames
104 live Ba Ames
10s live Ba Ames
10' live Ba Ames
10' heat-killed Ba Ames
102 live Ba Ames +
10' heat-killed Ba Ames
Average Ct*
BC3
NOT
37.4(1.5)
31.9(1.4)
29.3 (1.2)
28.6 (0.8)
25.9(1.5)
NOT
36.1 (0.3)
EPA-1
NOT
35.1(1.7)
30.8(0.5)
28.5(1.7)
28.0 (2.2)
24.6 (0.7)
NOT
33.8(1.8)
EPA-2
35.6(1.0)
29.2(1.4)
28.2(1.6)
24.5(1.3)
23.7 (0.7)
22.4 (2.7)
NOT
29.4(1.0)
*AverageCt(SD, n = 3)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units; NDT, no Ct detected
DNA extraction and purification results at T9 are
summarized in Table 44 (1:10 dilution performed to
insure reliable Ct values) and Table 45 (1:17 dilution
performed to equate volumes and concentrations
between TO and T9 aliquots and demonstrate sufficient
ACt to detect the presence of viable spores at T9). All
samples were detected using both 1:10 and 1:17 dilutions
at T9. Note that although the Ct values qualitatively
decrease with increasing concentrations of Ba spiked
in the samples, the Ct values are not separated by a full
log from one spore level to the other. However, overall
results are not affected because of an optimum ACt (Ct
change from TO to T9 at all spore levels).
Table 44. Manual RV-PCR at T9 on Wipes Processed with DNA Extraction and Purification Followed by 1:10
Dilution of Extracted Samples
CFU/Sample
10 live Ba Ames
102 live Ba Ames
103 live Ba Ames
104 live Ba Ames
10s live Ba Ames
10' live Ba Ames
10' heat-killed Ba Ames
102 live Ba Ames +
10' heat-killed Ba Ames
Average Ct*
BC3
32.7(1.0)
32.2 (2.0)
31.3(1.0)
29.7(0.2)
28.2(0.3)
27.3 (0.9)
NDT
33.9(1.5)
EPA-1
29.6(1.1)
28.5(1.2)
27.1(1.1)
26.2 (0.5)
23.7 (0.4)
23.4(0.3)
39.0(1.1)
30.0(1.2)
EPA-2
29.9(1.2)
27.1(0.8)
24.6 (0.7)
24.7 (0.9)
22.4 (0.4)
21.9(0.5)
38.7(0.9)
28.7(0.9)
Bg
31.4(1.3)
30.7(0.8)
31.2(1.6)
30.5 (0.6)
30.9(1.4)
30.3 (2.6)
32.2 (0.9)
31.0(0.7)
*Average Ct (SD, n = 3)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units; NDT, no Ct detected
-------
Table 45. Manual RV-PCR at T9 on 1:17 Diluted Purified DNAfrom Wipes (Table 41 Column 3)
CFU/Sample
10 live BaAines
102 live Ba Ames
103 live Ba Ames
104 live Ba Ames
10s live Ba Ames
10' live Ba Ames
10' heat-killed Ba Ames
102 live Ba Ames +
10' heat-killed Ba Ames
Average Ct*
BC3
36.6 (3.4)
35.7(2.9)
34.8(2.6)
30.9 (0.4)
31.0(2.8)
30.2 (2.4)
43.5(1.5)
35.6(2.5)
EPA-1
32.9 (2.4)
31.4(3.9)
31.4(2.3)
27.5 (0.4)
26.7(2.5)
26.2(2.5)
40.0(1.2)
32.6(1.6)
EPA-2
30.2(1.6)
28.8(2.5)
26.0 (2.6)
24.8(1.1)
23.8(1.5)
23.5(1.5)
36.1(1.2)
29.7(1.2)
Bg
32.3(2.1)
32.8 (2.7)
32.5 (2.5)
30.6 (0.3)
30.8 (2.4)
30.9(2.8)
32.3(0.1)
31.9(1.2)
*Average Ct (SD, n = 3)
Acronyms: Ct, cycle threshold; SD,
standard deviation; n, number of replicates; CPU, colony forming units
The Bg carrier DNA control at TO was not consistently
detected while consistent Ct values were measured on
the T9 samples. Table 46 shows the sensitivity of the
Bg assay with extracted Bg DNA, which is typically
5 fg per reaction. Based on these results, an additional
control experiment was performed in which TE and BHI
samples were spiked with a fixed concentration of Ba
spores and increasing concentrations of Bg DNA (see
Table 47).
Table 46. Ct Values Obtained with Extracted B.
globigii DNA Dilution Series
Bg DNA (pg)
5000
500
50
5
0.5
0.05
0.005
0
Average Ct*
15.8(0.1)
17.2(0.1)
20.3(0.1)
23.8(0.2)
27.1(0.1)
31.5(0.3)
35.6(0.3)
NOT
A final TO control experiment was then performed in
order to define the concentration of Bg carrier DNA to
spike in the TO samples as a positive control. In this
experiment, 2 sets of twelve 900 uL samples were
prepared (1 set with 900 uL of TE and 1 set with 900
uL of BHI). To these samples, 100 uL of Ba Ames
spore suspension at 104 CFU/mL were added, bringing
the final spore concentration to the 103 spore level in
each sample (the highest spiking concentration used
in this study). Samples were then spiked with various
concentrations of extracted Bg DNA in triplicate (final
Bg DNA concentrations of 50, 250, 500 and 750 pg/
mL were used). All twenty four 1 mL samples were
then processed with the Promega DNA extraction and
purification protocol and analyzed for the presence of Bg
DNAbyPCR.
Results presented in Table 47 clearly show that TE
interferes with the binding chemistry of the Promega
DNA extraction and purification protocol since no Ct
values were detected. Using similar concentrations of
Bg DNA in BHI samples, Bg was reliably detected for
spiking concentrations above 500 pg/mL.
*AverageCt(SD, n = 3)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of
replicates; NOT, no Ct detected
-------
Table 47. Ct values for 1 mL Samples Spiked with 103
Bacillus anthrads CFU/mL and B. globigii DNA, and
Processed with DNA Extraction and Purification
Sample
Matrix
TE
TE
TE
TE
BHI
BHI
BHI
BHI
Final Concentration of
Bg DNA (pg/mL)
50
250
500
750
50
250
500
750
Bg Assay*
NOT
NOT
NOT
NOT
NOT
30.8(0.6)**
30.4(1.0)
29.5(1.0)
*Average Ct (SD, n = 3)
**Ct values for 3 replicates were: 30.3, NDT, 31.2
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number
of replicates; CPU, colony forming units; pg, picogram; NDT, no Ct
detected
Based on the results of these TO control experiments,
final manual and semi-automated RV-PCR protocols
were designed for the analysis of real-world samples.
The detailed protocols in Appendices C and D and an
overall summary are provided in Table 48.
Table 48. Summary of Aliquot Equivalency between
TO Controls and T9 Samples for the Processing of
Real-World Samples
TO
100 uL sample + 900 uLBHI
(Total sample volume is 1 mL)
DNA extraction and purification
No dilution
PCR
T9
1 mL sample
DNA extraction and
purification
1:10 dilution
PCR
4.4. Single Laboratory Method
Verification
Based on the optimization experiments presented above,
a decision was made to proceed with the optimized
RV-PCR method (10 min of vortexing of the filter cups
prior to aliquoting the sample and 9 hr of incubation
followed by magnetic bead-based DNA extraction and
purification/extraction and purification prior to running
PCR) for the method verification phase of the project.
Both manual and semi-automated methods were verified
with a total of 192 wipe, air filter and water samples
spiked with Ba Ames spores. Dirt (wipes containing
250 mg of Arizona test dust, dirty Bio Watch filters and
chemically spiked water), high levels of heat-killed
Ba spores and high levels of non-target cells were also
evaluated. Four replicates of each sample were analyzed.
For each replicate sample, PCR was run against each of
the three assays (one PCR per sample and per assay).
All experiments were conducted according to manual
and semi-automated RV-PCR protocols detailed in
Appendices A and B.
4.4.7. Verification of the Manual RV-PCR Method
Clean wipes, air filters and water samples were spiked
at the 10 and 100 live Ba Ames spore levels and four
replicates were analyzed for each sample. Results are
presented in Tables 49-51 below.
The Ct values at T9 obtained during the verification of
the manual method for clean samples were < 35.0 for all
samples, confirming the 10 spore detection level with the
manual RV-PCR method.
Acronyms: uL, microliter
-------
Table 49. Average Ct Values for Manual RV-PCR at T9 on Clean Wipes with 2 Spore Levels
CFU/Sample
31
272
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
26.7(2.5)
45
20.8(1.5)
ACt**
18.3
24.2
EPA-2
Ct*1
45
27.0 (2.7)
45
20.1(0.6)
ACt**
18.0
24.9
BC3
Ct*1
45
24.6 (3.0)
45
22.8(0.6)
ACt**
20.4
22.2
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
Table 50. Average Ct Values for Manual RV-PCR at T9 on Clean Filters with 2 Spore Levels
CFU/Sample
31
272
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
30.5 (2.4)
45
21.8(1.1)
ACt**
14.5
23.2
EPA-2
Ct*1
45
31.0(2.4)
45
20.1 (0.5)
ACt**
14.0
24.9
BC3
Ct*1
45
27.4(2.1)
45
23.6(0.8)
ACt**
17.6
21.4
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
Table 51. Average Ct Values for Manual RV-PCR at T9 on Clean Water with 2 Spore Levels
CFU/Sample
31
272
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
33.7(1.8)
45
21.3 (0.7)
ACt**
11.3
23.7
EPA-2
Ct*1
45
32.4(1.5)
45
18.9(1.1)
ACt**
12.6
26.1
BC3
Ct*1
45
31.2(2.1)
45
23.4(1.0)
ACt**
13.8
21.6
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
-------
Dirty wipes (250 mg of Arizona test dust), dirty air
niters (BioWatch air niters collected in Houston, Texas
on 02/20/08) and dirty water (10 mg/L humic acid and
10 mg/L ferrous sulfate) samples were spiked at the 10
and 100 live Ba Ames spore level and four replicates
were analyzed for each sample. Results are presented in
Tables 52-54 below.
The Ct values at T9 obtained during the manual method
verification on dirty samples were < 36.0 for all samples,
confirming the 10 spore detection level in the presence
of dirt with the manual RV-PCR method.
Table 52. Average Ct Values for Manual RV-PCR at T9 on Dirty Wipes with 2 Spore Levels
CFU/Sample
49
301
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
34.6 (0.4)
45
31.4(1.8)
ACt**
10.4
13.6
EPA-2
Ct*1
45
33.2(1.3)
45
27.8(1.7
ACt**
11.8
17.2
BC3
Ct*1
45
35.6(1.8)
45
34.2(2.1)
ACt**
9.4
10.8
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
Table 53. Average Ct Values for Manual RV-PCR at T9 on Dirty Filters with 2 Spore Levels
CFU/Sample
49
301
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
34.6 (2.8)
45
31.7(0.9)
ACt**
10.4
13.3
EPA-2
Ct*1
45
33.5(1.7)
45
28.4(1.5)
ACt**
11.5
16.6
BC3
Ct*1
45
36.0(1.3)
45
34.0(1.0)
ACt**
9.0
11.0
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
Table 54. Average Ct Values for Manual RV-PCR at T9 on Dirty Water with 2 Spore Levels
CFU/Sample
49
301
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
33.8(0.5)
45
30.9 (0.9)
ACt**
11.2
14.1
EPA-2
Ct*1
45
31.6(0.4)
45
29.9(1.4)
ACt**
13.4
15.1
BC3
Ct*1
45
34.9(1.0)
45
34.1 (1.2)
ACt**
10.1
10.9
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
-------
Clean wipes, air niters and water samples were spiked
at the 10 and 100 live Ba Ames spore level in the
presence of 106 heat-killed Ba Ames spores/sample. Four
replicates were analyzed for each sample. Results are
presented in Tables 55-57 below.
The Ct values at T9 obtained during the manual method
verification in the presence of heat-killed Ba Ames
spores were < 35.0 for all samples, confirming the 10
spore detection level in the presence of a high level of
dead Ba Ames spores with the manual RV-PCR method.
Table 55. Average Ct Values for Manual RV-PCR at T9 on Clean Wipes with 2 Spore Levels in the Presence of
Heat-Killed Bacillus anthracis Spore Background
CFU/Sample
14
143
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
29.5(1.1)
45
28.8(0.4)
ACt**
15.5
16.2
EPA-2
Ct*1
45
26.1 (0.7)
45
26.1(0.2)
ACt**
18.9
18.9
BC3
Ct*1
45
32.8(0.7)
45
33.1(0.5)
ACt**
12.2
11.9
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
Table 56. Average Ct Values for Manual RV-PCR at T9 on Clean Filters with 2 Spore Levels in the Presence of
Heat-Killed Bacillus anthracis Spore Background
CFU/Sample
14
143
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
31.3(2.2)
45
27.1 (1.9)
ACt**
13.7
17.9
EPA-2
Ct*1
45
28.2 (2.4)
45
23.8(0.6)
ACt**
16.8
21.2
BC3
Ct*1
45
34.9 (0.4)
45
30.8(1.2)
ACt**
10.1
14.2
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
-------
Table 57. Average Ct Values for Manual RV-PCR at T9 on Clean Water with 2 Spore Levels in the Presence of
Heat-Killed Bacillus anthrads Spore Background
CFU/Sample
14
143
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
33.6(1.6)
45
31.4(1.8)
ACt**
11.4
13.6
EPA-2
Ct*1
45
28.7(1.0)
45
26.8(1.0)
ACt**
16.3
18.2
BC3
Ct*1
45
34.9(1.5)
45
34.8 (0.9)
ACt**
10.1
10.2
* Ct = Average Ct (SD, n = 4)
!Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
Manual RV-PCR method verification with clean
samples and a high live non-target background was then
performed. Clean wipes, air filters and water samples
were spiked at the 10 and 100 live Ba Ames spore level
in the presence of 106 live Pa CPU/sample and 103 live
Bg CPU/sample. Four replicates were analyzed for each
sample. Results are presented in Tables 58-60 below.
The Ct values at T9 obtained during the manual method
verification in the presence of live non-target background
were < 36.0 for all samples, confirming the 10 spore
detection level in the presence of high levels of live non-
target organisms with the manual RV-PCR method.
Table 58. Average Ct Values for Manual RV-PCR at T9 on Clean Wipes with 2 Spore Levels in the Presence of
live P. aeruginosa and B. globigii Background
CFU/Sample
33
186
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
31.6(2.7)
45
29.7(3.0)
ACt**
13.4
15.3
EPA-2
Ct*1
45
25.4(0.8)
45
21.2(0.9)
ACt**
19.6
23.8
BC3
Ct*1
45
33.5(1.6)
45
30.3(3.1)
ACt**
11.5
14.7
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
-------
Table 59. Average Ct Values for Manual RV-PCR at T9 on Clean Filters with 2 Spore Levels in the Presence of
live P. aeruginosa and B. globigii Background
CFU/Sample
33
186
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
32.5(1.8)
45
30.1 (3.5)
ACt**
12.5
14.9
EPA-2
Ct*1
45
26.3 (0.6)
45
23.4(2.0)
ACt**
18.7
21.6
BC3
Ct*1
45
33.8(3.8)
45
32.0 (3.0)
ACt**
11.2
13.0
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
Table 60. Average Ct Values for Manual RV-PCR at T9 on Clean Water with 2 Spore Levels in the Presence of
live P. aeruginosa and B. globigii Background
CFU/Sample
33
186
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
34.3 (4.5)
45
31.3(3.4)
ACt**
10.7
13.7
EPA-2
Ct*1
45
29.9 (0.6)
45
26.7(2.1)
ACt**
15.1
18.3
BC3
Ct*1
45
35.3 (3.8)
45
29.4(1.7)
ACt**
9.7
15.6
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
4.4.2. Verification of the Semi-Automated
RV-PCR Method
Clean wipes, air niters and water samples were spiked
at the 10 and 100 live Ba Ames spore levels and four
replicates were analyzed for each sample. Results are
presented in Tables 61-63 below.
The Ct values at T9 obtained during the semi-automated
method verification on clean samples were < 35.0 for all
samples, confirming the 10 spore detection level with the
semi-automated RV-PCR method.
-------
Table 61. Average Ct Values for Semi-automated RV-PCR at T9 on Clean Wipes with 2 Spore Levels
CFU/Sample
26
203
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
28.9(1.1)
45
25.0(1.0)
ACt**
16.1
20.0
EPA-2
Ct*1
45
26.5 (0.3)
45
23.3(1.6)
ACt**
18.5
21.7
BC3
Ct*1
45
31.9(1.0)
45
28.1 (2.6)
ACt**
13.1
16.9
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
Table 62. Average Ct Values for Semi-automated RV-PCR at T9 on Clean Filters with 2 Spore Levels
CFU/Sample
26
203
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
30.8(2.1)
45
26.4 (2.9)
ACt**
14.2
18.6
EPA-2
Ct*1
45
28.7(2.2)
45
25.3 (2.6)
ACt**
16.3
19.7
BC3
Ct*1
45
33.2(3.5)
45
33.8(1.7)
ACt**
11.8
11.2
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
Table 63. Average Ct Values for Semi-automated RV-PCR at T9 on Clean Water with 2 Spore Levels
CFU/Sample
26
203
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
29.0(1.9)
45
26.8(1.3)
ACt**
16.0
18.2
EPA-2
Ct*1
45
27.6 (2.0)
45
25.9(1.8)
ACt**
17.4
19.1
BC3
Ct*1
45
33.2(2.0)
45
31.4(2.2)
ACt**
11.8
13.6
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
-------
A replicate manifold of clean samples was spiked at
the 10 and 100 live Ba spore levels, in order to plate
samples at TO and T9. This manifold was processed in
a similar manner as the previous manifold. Filter cups
were vortexed for 10 min at TO and a 100 |j,L aliquot
was plated out of each filter cups (24 plates). After 9
hr of incubation, filter cups were vortexed for 10 min
and serial dilutions were prepared in BHI and plated
in triplicate for each sample (216 plates). Results are
provided in Table 64.
No live organism was detected by plating at TO for the
10 spore level (10 spores spiked in the sample become
2.6 CFU/mL in the filter cup assuming 100% recovery,
which then becomes 0.26 spores/100 uL for plating).
For the 100 spore level (100 spores spiked in the sample
become 26 CFU/mL in the filter cup assuming 100%
recovery which then becomes 2.6 spores/100 uL for
plating), colonies were detected most of the time (the
number of colonies per plate was below 10, as expected).
These results exemplify the advantage of the RV-PCR
method over the plating method for the detection of low
levels of live Ba spores. After 9 hr of incubation in filter
cups, the cell concentration was 106 cells/filter cup when
starting with a spiking level of 10 CPU/sample, and 107
cells/filter cup when starting with a spiking level of 100
CPU/sample. Such growth corresponds to a doubling
time in the order of 30 min and a germination time in
the 30-60 min range, which suggests optimal growth
conditions for Ba in filter cups and confirms that the Ct
values recorded at T9 originate from viable spores.
Table 64. Spore Recovery Experiment from Filter Cup Aliquots at TO and T9
Sample Type
Clean Filter
Clean Water
Clean Wipe
Clean Filter
Clean Water
Clean Wipe
Ba Ames Spore
Level
10
10
10
100
100
100
Plate Counts
™TT ., ,, , CPU/filter cup CPU/filter cup
CFU spiked/sample ^ p T9**
29
29
29
29
29
29
29
29
29
29
29
29
262
262
262
262
262
262
262
262
262
262
262
262
0
0
0
0
0
0
0
0
0
0
0
20
120
60
0
20
40
20
0
20
20
60
0
0
1,720,000
1,020,000
1,560,000
1,340,000
1,000,000
840,000
820,000
840,000
1,760,000
1,940,000
1,080,000
2,020,000
12,600,000
11,200,000
10,200,000
7,200,000
8,000,000
7,400,000
7,800,000
5,800,000
28,400,000
28,800,000
24,600,000
21,200,000
*One 100 uL aliquot was plated out of each filter cup at TO. Number of spores in filter cup = number of colonies x 10 x 2.
* * Serial 10-fold dilutions were prepared out of each filter cup and plated in triplicate. The third, fourth and fifth dilutions were plated for the
samples spiked at the 10 spore level. The fourth, fifth and sixth dilutions were plated for the samples spiked at the 100 spore level. Values presented
in the table are corrected from dilution. Each value is the average of three replicate plates.
Acronyms: CFU, colony forming unit
-------
Dirty wipes (250 mg of Arizona test dust), air niters
(BioWatch air niters collected in Houston, Texas on
02/20/08) and water (10 mg/L humic acid and 10 mg/L
ferrous sulfate) samples were spiked at the 10 and
100 live Ba Ames spore level and four replicates were
analyzed for each sample. Results are presented in
Tables 65-67 below.
The Ct values at T9 obtained during the semi-automated
method verification on dirty samples were < 35.0 for
all samples, confirming the 10 spore detection level in
the presence of dirt with the semi-automated RV-PCR
method.
Table 65. Average Ct Values for Semi-automated RV-PCR at T9 on Dirty Wipes with 2 Spore Levels
CFU/Sample
40
327
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
32.1(7.7)
45
26.0 (3.6)
ACt**
12.9
19.0
EPA-2
Ct*1
45
28.5 (4.9)
45
23.5 (3.2)
ACt**
16.5
21.5
BC3
Ct*1
45
30.5(1.8)
45
27.6 (3.3)
ACt**
14.5
17.4
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
Table 66. Average Ct Values for Semi-automated RV-PCR at T9 on Dirty Filters with 2 Spore Levels
CFU/Sample
40
327
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
32.3 (2.3)
45
31.9(1.8)
ACt**
12.7
13.1
EPA-2
Ct*1
45
30.4(2.1)
45
29.8(1.6)
ACt**
17.6
15.2
BC3
Ct*1
45
34.6 (3.0)
45
34.8(1.4)
ACt**
10.4
10.2
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
Table 67. Average Ct Values for Semi-automated RV-PCR at T9 on Dirty Water with 2 Spore Levels
CFU/Sample
40
327
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
29.7(2.3)
45
26.8 (0.3)
ACt**
15.3
18.2
EPA-2
Ct*1
45
28.2(2.5)
45
24.9 (0.3)
ACt**
16.8
20.1
BC3
Ct*1
45
32.0 (2.2)
45
29.0 (0.9)
ACt**
13.0
16.0
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
-------
Clean wipes, air niters and water samples were spiked
at the 10 and 100 live Ba Ames spore level in the
presence of 106 heat-killed Ba Ames spores/sample. Four
replicates were analyzed for each sample. Results are
presented in Tables 68-70 below.
The Ct values at T9 obtained during the semi-automated
verification in the presence of heat-killed Ba Ames
spores were < 35.0 for all samples, confirming the 10
spore detection level in the presence of a high level
of dead Ba Ames spores with the semi-automated RV-
PCR method. These results directly relate to real-world
situations since the presence of high levels of dead Ba
spores will be encountered in post-decontamination
samples.
Table 68. Average Ct Values for Semi-automated RV-PCR at T9 on Clean Wipes with 2 Spore Levels in the
Presence of Heat-Killed Bacillus anthracis Spore Background
CFU/Sample
38
256
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
31.8(1.2)
45
30.3(1.0)
ACt**
13.2
14.7
EPA-2
Ct*1
45
29.2(1.2)
45
28.0(1.6)
ACt**
15.8
17.0
BC3
Ct*1
45
32.7(1.8)
45
32.0(1.8)
ACt**
12.3
13.0
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
Table 69. Average Ct Values for Semi-automated RV-PCR at T9 on Clean Filters with 2 Spore Levels in the
Presence of Heat-Killed Bacillus anthracis Spore Background
CFU/Sample
38
256
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
26.5(1.8)
45
25.9(1.4)
ACt**
18.5
19.1
EPA-2
Ct*1
45
24.7(1.5)
45
23.9(1.0)
ACt**
20.3
21.1
BC3
Ct*1
45
29.0(1.8)
45
28.5(1.2)
ACt**
16.0
16.5
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
Table 70. Average Ct Values for Semi-automated RV-PCR at T9 on Clean Water with 2 Spore Levels in the
Presence of Heat-Killed Bacillus anthracis Spore Background
CFU/Sample
38
256
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
30.3 (3.2)
45
26.8(1.0)
ACt**
14.7
18.2
EPA-2
Ct*1
45
28.1 (3.0)
45
24.5 (0.6)
ACt**
16.9
20.5
BC3
Ct*1
45
30.6 (0.7)
45
28.4(1.0)
ACt**
14.4
16.6
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
-------
Semi-automated RV-PCR verification with clean
samples and a high live non-target background was then
performed. Clean wipes, air filters and water samples
were spiked at the 10 and 100 live Ba Ames spore level
in the presence of 106 live Pa CPU/sample and 103 live
Bg CPU/sample. Four replicates were analyzed for each
sample. Results are presented in Tables 71-73 below.
The Ct values at T9 obtained during the semi-automated
verification in the presence of live non-target background
were < 35.5 for all samples, confirming the 10 spore
detection level in the presence of high levels of live
non-target organisms with the semi-automated RV-PCR
method.
Table 71. Average Ct Values for Semi-automated RV-PCR at T9 on Clean Wipes with 2 Spore Levels in the
Presence of P. aeruginosa and B. globigii Background
CFU/Sample
21
233
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
27.2 (0.4)
45
26.8 (0.4)
ACt**
17.8
18.2
EPA-2
Ct*1
45
24.8(0.5)
45
24.8 (0.6)
ACt**
20.2
20.2
BC3
Ct*1
45
31.3(0.7)
45
31.0(1.0)
ACt**
13.7
14.0
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
Table 72. Average Ct Values for Semi-automated RV-PCR at T9 on Clean Filters with 2 Spore Levels in the
Presence of P. aeruginosa and B. globigii Background
CFU/Sample
21
233
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
31.6(1.3)
45
29.5(1.5)
ACt**
13.4
15.5
EPA-2
Ct*1
45
28.5 (0.8)
45
27.5(1.6)
ACt**
16.5
17.5
BC3
Ct*1
45
35.2(0.5)
45
34.0(1.8)
ACt**
9.8
11.0
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
Table 73. Average Ct Values for Semi-automated RV-PCR at T9 on Clean Water with 2 Spore Levels in
the Presence of P. aeruginosa and B. globigii Background
CFU/Sample
21
233
Time
Point
TO
T9
TO
T9
Assay
EPA-1
Ct*1
45
28.5 (0.3)
45
28.2 (0.3)
ACt**
16.5
16.8
EPA-2
Ct*1
45
26.6 (0.2)
45
26.3 (0.3)
ACt**
18.4
18.7
BC3
Ct*1
45
32.8(0.3)
45
32.8(0.3)
ACt**
12.2
12.2
* Ct = Average Ct (SD, n = 4)
1 Ct values at TO were all 'non-detect'. A Ct value of 45 was entered in the table in order to calculate the ACt value.
**ACt=Ct(TO)-Ct(T9)
Acronyms: Ct, cycle threshold; SD, standard deviation; n, number of replicates; CPU, colony forming units
-------
To summarize Section 4, single laboratory verification
of the RV-PCR method was conducted for wipes, air
filters and water samples for both manual and semi-
automated protocols, using 9 hr of incubation followed
by DNA extraction and purification and PCR. Spore
levels of 10 and 100 CPU were tested and 4 replicates
were processed for each sample type/spore level/
challenge combination. Challenges introduced during
testing included the addition of dirt or debris, high levels
of heat-killed Ba spores and high levels of live non-
target organisms. Out of 192 positive samples analyzed
during verification, 188 samples presented Ct values <
35 for all 3 assays (chromosome, and pXOl and pXO2
plasmids). For the 4 samples outside this range, the 2
plasmid assays had Ct values < 35 and the chromosome
assay Ct values were 35.6 (manual, with dirt). 36.0
(manual, with dirt). 35.3 (manual, with live background)
and 35.2 (semi-automated, with live background)
respectively. Detection at 10 spore level was maintained
during the verification of both the manual and semi-
automated version of the Ba RV-PCR method.
-------
-------
5.0
Conclusions
Single laboratory verification of both manual and semi-
automated versions of this optimized method showed
limits of detection at the 10 spore level with and without
debris for all three sample types. Live Ba Ames spores
were consistently detected at the 10 spore level for both
manual and semi-automated methods in heat-killed
Ba spore backgrounds of 106 spores/sample and live,
combined non-target backgrounds of 103 Bg and 106
Pa. The method endpoint was shortened from its initial
overnight incubation (16 hr) to 9 hr by performing a
magnetic bead-based DNA extraction and purification
procedure before PCR analysis. Using this method, the
total manual processing time from start to finish for 24
samples was reduced to 14-15 hr (3-4 hr of processing
time should be added for each set of 24 samples).
Additional work is needed however. This research
did not focus on sampling methods and no exhaustive
list of potential growth and/or PCR inhibitors was
assessed. Although dirt was added to wipes, no actual
surface wiping was performed to test interferences
from chemicals and/or debris from sampled surfaces.
Although air filter samples were randomly collected
from subway and outdoor locations, seasonal variations
such as high versus low pollen levels or high versus
low air pollution levels were not systematically tested.
Although water samples were spiked with humic acid
and ferrous oxide, which arc known PCR inhibitors,
no environmental samples were tested in this study.
Interference of the decontamination method (fumigation.
foam) with the RV-PCR method was not examined
although the typical effects of decontamination are
delayed germination, growth, and PCR inhibition.
Experiments addressing these additional issues were
outside the scope of this study since virulent Ba was
used.
In addition, the RV-PCR method described here was
developed for qualitative and not quantitative analysis.
Although die results showed a qualitative decrease
in Ct values with increasing spore spiking levels,
the Ct difference between two consecutive 10-fold
dilutions was typically below the theoretical value in
the presence of challenges, which suggests that the
lack of quantitation of die method may be tied to the
environmental component of the samples (dirt, dead
spore background, live non-target background). Follow
on work will be performed in FY10 to determine the
relationship between limit of detection and incubation
time, using clean wipe samples. These experiments will
evaluate whether the method endpoint could be further
reduced when processing relatively clean samples such
as wipes collected from clean indoor locations.
Although the main focus of the NHSRC-funded effort
was on delivering verified, rapid viability test protocols
to support ERLN capabilities to ensure public safety,
other potential applications for the RV-PCR method may
lie in surveillance, public health and food safety.
-------
-------
This work was performed under the auspices of the
U.S. Department of Energy by Lawrence Livermore
National Laboratory (LLNL) under Contract DE-AC52-
07NA27344. Funding for this research was provided by
the US Environmental Protection Agency.
We thank Cheryl Strout at LLNL for the help that she
provided with the Ba assay selectivity study. We are also
grateful to Tom Slezak and Pejman Naraghi-arani, also at
LLNL. for providing us access to the sequence of assays
originally developed in their groups. These assays were
screened in our bio-informatics down-selection task.
During many stages of this collaborative Interagency
Agreement project, significant technical input was
provided by Dr. Sanjiv Shah of the U.S. EPAs NHSRC.
6.0
Acknowledgments
-------
-------
7.0
References
1. S. Kane, S. Letant G. Murphy, T. Alfaro, P. Krauter,
R. Mahnke, T. Legler and E. Raber. 2009. Rapid,
high-throughput, culture-based PCR methods
to analyze samples for viable spores of Bacillus
anthracis and its surrogates. J. Microbiol. Methods
76(3), 278-284.
2. S. Letant S. Kane, G. Murphy, T. Alfaro, L. Hodges,
L. Rose and E. Raber. 2010. Most-probable-number
rapid viability PCR method to detect viable spores
of Bacillus anthracis in swab sample.«/. Microbiol.
Methods 81(2), 200-202.
3. S. Kane, S. Letant, T. Mitchell-Hall, T. Bunt, and
E. Raber. 2008. Quality Assurance Project Plan:
Development and Verification of Rapid Viability
Polymerase Chain Reaction (RV-PCR) Protocols for
Bacillus anthracis - For Application to Air Filter,
Water and Surface Samples: for EPA under IAG
#DW-89-9226]60l-O.LLNL-TM-406527.
4. Materials Safety Data Sheet. Arizona Test Dust,
2006. Powder Technology Inc., 14331 Ewing
Avenue S., Burnsville. MN 55306. http://www.
powdertechnologyinc.com/products/test-dust/
arizona-test-dustphp
5. L. Hodges, L. Rose, H. O'Cornell and M. Arduino.
National validation study for a swab protocol for the
recovery of Bacillus anthracis spores from surfaces.
Poster session presented at: American Society for
Microbiology (ASM) Biodefense and Emerging
Diseases Research Conference, February 22-25,
2009, Baltimore, Maryland.
6. Promega Technical Bulletin TB312. MagneSH(R)
blood genomic, max yield system. 2009. http://www.
promega.com/tbs/tb312/tb312.pdf
7. E. Bode, W. Hurtle, and D. Norwood. 2004.
Real-Time PCR assay for a unique chromosomal
sequence of Bacillus anthracis. J. Clin. Microbiol.
42(12), 5825-5831.
-------
-------
Appendix A:
Manual RV-PCR Protocol
Qualitative of in wipe, air filter
Appendix E, buffers and media; Appendix F, PCR Conditions; and Appendix G, Consumables
Laboratory set-up
* Put PPE (personal protection equipment) on: lab coat, safety glasses, double gloves.
• Prepare fresh bleach solution (1 volume bleach + 9 volumes water). Date and label with initials.
• Clean/bleach BioSafety Cabinet (BSC) and bench surfaces.
* All sample manipulations are perfonned in the BSC
Place samples in
* Wipes and air filters: Using sterile forceps, transfer sample collection type to 30 inL tube behind internal mesh/
support (1.5 mL of IX wash buffer is added to each wipe prior to spiking).
* Water samples: No mesh support is needed. Aliquot 20 mL of water directly in 30 mL tube.
Preparation of the working for sample spiking
Using Biological Safety Level 2 (BSL2) practices and BSL 3 pipetting techniques, start the protocol below.
1. Place original Ba Ames (108 CFU/mL; check vial for correct concentration) spore suspension on platform
vortexer for 20 min using position 7 (high).
2. Transfer stock to BSC.
3. Aliquot 100 \,iL of spore suspension into 900 \\L of IX wash buffer (working suspension of 107 CFU/mL).
4. Vortex on single tube vortexer for 1 min.
5. Aliquot 500 \\L of the working suspension into 4.5 mL of IX wash buffer (106 CFU/mL).
6. Vortex on single tube vortexer for 1 min.
7. Aliquot 3 mL of 106 CFU/mL solution to 27 mL of IX wash buffer (105 CFU/mL).
8. Vortex on single tube vortexer for 1 min.
9. Aliquot 1 mL of 10s CFU/mL to 9 mL of IX wash buffer (10" CFU/mL).
10. Aliquot 1 mL of 104 CFU/mL to 9 mL of IX wash buffer (10' CFU/mL).
11. Aliquot 1 mL of 103 CFU/mL to 9 mL of IX wash buffer (102 CFU/mL).
12. Plate on BHI agar plates as follows:
100 nL 102 CFU/mL (3 replicates)
50 nL 103 CFU/mL (3 replicates)
13. Incubate agar plates at 30°C overnight; count plates the next day to determine exact number of spores spiked in
the samples.
14. Inoculate sample tubes with 100 (oL of 102 CFU/mL (10 spore level) and 103 CFU/mL (100 spore level); record
manifold layout.
-------
Preparation ofBioSafety Cabinet for manual RV-PCR
Materials:
• Bleach wipes and daily bleach bottle
• Sharps waste container
« Absorbent pad
* Beaker with top filter-cup caps
• Capping tray with bottom caps
• 2 large biohazard bags + rubber bands
• Allen wrench for manifold
« Serological pipettes and pipettor
Setup:
• Set up manifold: connect vacuum pump, pump in line filter, and waste container with fresh bleach; tape pump
exhaust tube to BSC to vent exhaust inside BSC
* Add filter cups to manifold
• Tape filter-cup layout on glass window of the BSC
* Tape PCR plate layout on glass window of the BSC
Preparation of the for manual RW-PCR
Materials:
* Gloves
* Bleach wipes
* Waste bags
• Absorbent pads
• Isopropyl alcohol squeeze bottle
« Deionized water squeeze bottle
* Red bio hazards bags + rubber bands
• Autoclave tape
• Large photo-tray
• Sharps container
« Zip-lock bag containing: 96-well plate, plate support, foil seals, and plate sealer
* 2 mL eppendorf tubes
• 200 |j.L pipettor and pipette tips
* Marker
Media and Buffers (See Appendix E):
• Brain Heart Infusion medium
« High salt wash buffer (pH 6.0)
• IX wash buffer (pH 7.4)
-------
Manual protocol (TO)
1. IntheBSC, add 20 mL of extraction buffer to wipe or air filter samples placed in a 30 mL tubes (no addition is
needed for water samples).
2. Seal sample rack in large waste bag; bleach exterior of bag with bleach wipe.
3. Vortex samples for 20 min on platform vortexer (outside BSC), position 7.
4. After vortexing samples, transfer sample rack to BSC. Remove tube rack from plastic bag.
5. Turn on vacuum pump at 10 psi.
6. Using BSL3 pipetting technique, transfer sample from 30 mL sample tube to filter-cup using a serological
pipette (the total sample volume is 20 mL. Pipette 15 mL and deliver 13 mL to filter-cup). Dispose pipette
in waste container. Cap sample tube, bleach the outer surface of sample tube. Re-place tube back on rack.
Change gloves.
7. Repeat step 6 for each sample.
8. Complete filtration of liquid through filter cups.
9. Bleach sample tube rack. Double-bag rack. Bleach outer surface of bioliazard bag. Change gloves.
10. Transfer double-bagged sample tube rack to refrigerator.
11. Transfer 7 mL of high salt wash buffer (pH 6.0) to each filter-cup using a serological pipette. Use new
disposable pipette for each sample cup.
12. Complete filtration of liquid through filter cups.
13. Transfer 3 mL of IX wash buffer (pH 7.4) to each filter-cup using a serological pipette. Use new disposable
pipette for each sample cup.
14. Complete filtration of liquid through filter cups. Change gloves.
15. Stop vacuum pump.
16. Bring 5 mL graduated pipettes, 200 \,iL pipettor, 200 \,iL tips, BHI medium, sharps container, centrifuge safety
cup. zip-lock bag with PCR plate, plate support, foil seals, and plate sealer to BSC. Set up work space in BSC.
Change gloves.
17. Pipette 2.5 mL of Brain Heart Infusion medium into each filter cup using a serological pipette. Use new
disposable pipette for each filter cup.
18. Transfer upper part of manifold to capping tray fitted with bottom caps. Press down to cap bottom of filter-
cups. Change gloves.
19. When performing standard RV-PCR protocol (16 far incubation):
* Take 96-well PCR plate and plate support out of zip-lock bag.
* For each sample: swirl pipette tip gently in filter cup to mix sample, aliquot 60 (oL volume out of each cup
and transfer 50 j,iL to the 96-well plate (TO sample plate) according to plate layout (see example at the end of
this protocol).
• Seal TO sample plate with foil seal. Change gloves.
* Place TO 96-well plate in thermocycler for lysis: 20 minutes at 95°C. Once cycle is completed and sample is
back at room temperature, bag 96-well plate in zip-lock bag, bleach the bag.
• Transfer bagged 96-wcll plate to freezer and store at -20°C until qPCR is run (use a photo-tray to transport
plate).
When performing the optimized RV-PCR protocol (9 hr incubation and Promega DNA extraction and
purification protocol):
• For 3 samples (3 replicate samples spiked at a level of 102 or 103): swirl pipette tip gently in filter cup to mix
sample, aliquot 120 ^L volume out of each cup using and transfer 100 \iL to 2 mL eppendorf tubes.
* Add 900 (oL of BHI to each eppendorf tube.
* Add 500 pg of extracted Bg DNA to each eppendorf tube.
• Process 1 mL samples according to Promega MD1630 protocol described below.
20. Cap filter-cups. Double-bag filter-cup manifold. Bleach bag.
-------
21. Transfer bagged filter-cup tray to shaker incubator at 37°C, speed 230 rpm. Incubate for 16 br for the
standard RV-PCR protocol or for 9 hr for the optimized RV-PCR protocol.
22. Follow laboratory* cleanup protocol in Appendix E.
Manual RV-PCR protocol (T18 for standard protocol)
I. After incubation, take fi Iter-cup manifold out of incubator.
2. Vortex filter cups for 10 iriin on platform vortexer.
3. Transfer filter-cup manifold to BSC.
4. Using BSL3 pipetting technique, aliquot 60 |j.L volume out of each cup and transfer 50 |j.L to 96-wcll plate
using a micropipette (T16 sample plate) according to plate layout.
5. Seal Tl 6 plate with foil seal.
6. Lyse sample plate for 20 minutes at 95°C on thermocycler.
7. Once lysis is complete and plate is at room temperature, bag 96-well plate in zip-lock bag, bleach the bag.
8. Transfer plate to freezer and store at -20°C until qPCR is run (use a photo-tray to transport plate).
Manual Promega MD163Q DNA Extraction Purification Protocol (T9 for optimized protocol)
1. After incubation, lake filter-cup manifold out of incubator.
2. Vortex filter cups for 10 min on platform vortexer.
3. Transfer filter-cup manifold to BSC.
DNA extraction and purification phase:
4. Aliquot 1000 jj,L volume out of each filter cup to 2 mL eppendorf tube using a micropipette (T9 samples). Do
not use the 1.5 mL eppendorf tubes.
5. 1st lysis: add 600 ^L of bead mix (lysis buffer + magnetic beads) to each tube and mix.
6. Add 360 (oL of lysis buffer to each tube. Mix, place on magnet, discard supernatant to waste.
7. 2nd lysis: add 360 |j,L of lysis buffer to each tube. Mix, place on magnet, discard supernatant to waste.
8. 1st salt wash: add 360 p.L of salt wash solution to each tube. Mix, place on magnet, discard supernatant to
waste. Repeat one more time.
9. 1st alcohol wash: add 360 jjJL of alcohol wash solution to each tube. Mix, place on magnet, discard
supernatant to waste. Repeat one more time.
10. Air dry (2 min)
11. Heat dry on heat block at 80°C until samples are dry. Allow aU alcohol solution to evaporate since alcohol
may interfere with qPCR.
DNA concentration and elution phase:
12. Move sample tubes out of heat block and add 200 \\L of elution buffer to each tube. Mix, place on magnet,
collect supernatant with micropipette and transfer to clean eppendorf tubes (typically. 80 j,iL are collected).
Visually verify absence of magnetic bead cany over during final transfer. If magnetic bead carry over
occurred, place tube back on magnet, collect supernatant, and transfer to clean tube.
13. Store DNA samples at 4 °C until qPCR is run (use photo-tray to transport sample tubes).
14. Follow laboratory cleanup protocol in Appendix E.
-------
PCR for standard RV-PCR protocol with 16 hr incubation (follow PCR conditions provided in
Appendix F) - Perform on TO T16
1. Set up PCR plate with PCR mix according to plate layout in PCR-preparation hood, seal and transfer to BSC.
2. Take 96-well sample crude lysate plate out of freezer (use a photo-tray to transport plate), transfer to BSC.
Change gloves. Crude lysate plate should be kept on a cool block or at 4 °C at all times.
3. Using BSL3 centrifugation technique (PCR plates are loaded in safety cups), centrifuge sealed sample plate
for 1 inin at 2000 rpm.
4. Open crude lysate plate in BSC.
5. Mix samples up and down 10 limes and transfer 5 |_iL from crude lysate plate to the PCR plate (with PCR
mix). Seal PCR plate with clear seal. Change gloves.
6. When working with dirt}' wipes, perform 1:10 dilution of crude lysate:
Add 90 jjL of PCR-grade water to wells of a sterile 96-well bioblock (See Appendix G).
Mix crude cell lysate up and down 5 times and transfer 10 j,iL to bioblock wells, maintaining the plate layout.
Mix diluted samples up and down 10 times and transfer 5 |j,L from bioblock well to the PCR plate (with PCR
mix). Seal PCR plate with clear seal. Change gloves.
7. Using BSL3 centrifugation technique, centrifuge sealed PCR plate for 1 min at 2000 rpm.
8. Open safety cup in BSC, place plate on photo-tray, change gloves, transfer PCR plate to AB1 thermocycler.
9. Rim PCR (Please see Appendix F).
10. After thermal cycling completion, discard sealed PCR plate to waste. Autoclave. PCR plates with amplified
product are never to be opened in the laboratory.
11. Follow laboratory cleanup protocol in Appendix E.
PCR for optimized RV-PCR protocol with 9 hr of incubation and Promega DNA extraction and
purification (follow PCR conditions provided in Appendix F) - Perform on TO and T9 samples
1. Set up PCR plate with PCR mix according to plate layout in PCR-preparation hood, seal, transfer to BSC.
2. If sample tubes were frozen, transfer them to BSC and let them thaw to room temperature.
3. For TO samples: mix samples up and down 10 times and transfer 5 uL from each tube to the PCR plate (with
PCR mix). Seal PCR plate with clear seal. Change gloves.
4. Perform 1:10 dilution of all T9 samples:
Add 90 p.L of PCR-grade water to wells of a sterile 96-well bioblock.
Mix sample up and down 5 times and transfer 10 uL to bioblock wells, maintaining the plate layout.
Mix diluted samples up and down 10 times and transfer 5 ^L from bioblock well to the PCR plate (with PCR
mix). Seal PCR plate with clear seal. Change gloves.
5. Using BSL3 centrifugation technique, centrifuge sealed PCR plate for I min at 2000 rpm.
6. Open safety cup in BSC, place plate on photo-tray, change gloves, transfer PCR plate to ABI thermocyclcr.
7. Rim PCR (Please see Appendix F).
8. After thermal cycling completion, discard sealed PCR plate to waste. Autoclave. PCR plates with amplified
product are never to be opened in the laboratory.
9. Follow laboratory cleanup protocol in Appendix E.
-------
Example of 24 sample manifold lay-out:
Sample Al
Sample Bl
Sample Cl
Sample Dl
Sample El
Sample Fl
Sample Gl
Sample HI
Sample A2
Sample B2
Sample C2
Sample D2
Sample E2
Sample F2
Sample G2
Sample H2
Sample A3
Sample B3
Sample C3
Sample D3
Sample E3
Sample F3
Sample G3
Sample H3
Corresponding PCR lysis plate lay-out:
Al
Bl
Cl
Dl
El
Fl
Gl
HI
A2
B2
C2
D2
E2
F2
G2
H2
A3
B3
C3
D3
E3
F3
G3
H3
-------
Appendix B:
Semi-automated RV-PCR Protocol
Qualitative of in wipe, air filter
Laboratory set-up
* Put PPE (personal protective equipment) on: lab coat, safer}' glasses, double gloves.
• Prepare fresh bleach solution (1 volume bleach + 9 volumes water). Date and label with initials.
* Clean/bleach BioSafety Cabinet (BSC), Janus enclosure and bench surfaces.
* All sample manipulations are performed in the BSC or in the HEPA-filtered Janus enclosure.
Place in
* Wipes and air filters: Using sterile forceps, transfer sample collection type to 30 mL tube behind internal mesh/
support (1.5 mL of IX wash buffer is added to each wipe prior to spiking).
• Water samples: No mesh support is needed. Aliquot 20 mL of water directly in 30 mL tube.
Preparation of the spore working for sample spiking
Using Biological Safety Level 2 (BSL2) practices and BSL3 pipetting techniques, start the following protocol:
1. Place original Ba Ames (10s CFU/mL; check vial for correct concentration) spore suspension on platform
vortexer for 20 min using position 7 (high).
2. Transfer stock to BSC
3. Aliquot 100 jjL of spore suspension into 900 (iL of IX wash buffer (working suspension of 107 CFU/mL)
4. Vortex on single tube vortexer for 1 min
5. Aliquot 500 joL of the working suspension into 4.5 mL of IX wash buffer (106 CFU/mL)
6. Vortex on single tube vortexer for 1 min
7. Aliquot 3 mL of JO6 CFU/mL solution to 27 mL of IX wash buffer (105 CFU/mL)
8. Vortex on single tube vortexer for 1 min
9. Aliquot 1 mL of 105 CFU/mL to 9 mL of IX wash buffer (104 CFU/mL)
10. Aliquot 1 mL of 104 CFU/mL to 9 mL of IX wash buffer (103 CFU/mL)
11. Aliquot 1 mLof 103 CFU/mL to 9 mL of IX wash buffer (102 CFU/mL)
12. Plate on BHI agar plates as follow:
100 (iL 102 CFU/mL (3 replicates)
50 p,L 103 CFU/mL (3 replicates)
13. Incubate agar plates at 30°C overnight; count plates the next day to determine exact number of spores spiked
in the samples.
14. Inoculate sample tubes with 100 \\L of 102 CFU/mL (10 spore level) and 103 CFU/mL (100 spore level); record
manifold layout.
-------
Preparation of the Janus Robotic enclosure
Materials:
• Sharps waste container to collect tips
• Beaker with top filter-cup caps
« Capping tray with bottom caps
* 2 large bio hazard bags + rubber bands
• Allen wrench for manifold
Setup:
* Bleach wipes and daily bleach bottle
• Set up manifold: connect vacuum pump, pump in line filter, and waste container with fresh bleach; tape pump
exhaust tube to Janus enclosure to vent exhaust inside enclosure
• Add filter cups to manifold
« Tape filter-cup layout on glass window of the enclosure
* Tape PCR plate layout on glass window of the enclosure
Preparation of the cart
Materials:
• Gloves
• Bleach wipes
* Waste bags
* Isopropyl alcohol squeeze bottle
* Deionized water squeeze bottle
• Red biohazards bags + rubber bands
• Autoclave tape
« Large photo-tray
* Reservoirs
• Sharps containers
• Robotic tip refills
• Zip-lock bag containing: 96-well plate, plate support, foil seals, and plate sealer
« 2 mLeppendorf tubes
* Pipettor and pipette tips: 200 |jL
• Marker
Media and Buffers:
* Brain Heart Infusion medium
• High salt wash buffer (pH 6.0)
« IX wash buffer (pH 7.4)
preparation in BSC
I. Using BSL3 pipetting technique, add 20 mL of extraction buffer to wipes and air filter samples placed in a 30
mL tubes (not needed for water samples).
2. Seal sample rack in waste plastic bag; bleach exterior of bag with bleach wipes.
3. Vortex samples for 20 min on platform vortexer, position 7 (outside BSC).
4. After vortexing samples, transfer sample rack to Robotic enclosure. Remove tube rack from plastic bag.
-------
Semi-automated RV-PCR (TO)
1. Turn on vacuum pump at 10 psi.
2. Using the Janus robotic system, transfer 13 mL from 30 mL sample tubes to filter-cups. Stop robot, cap
sample tubes, bleach the outer surface of sample tubes, bag the tube rack, bleach the bag. and transfer to
refrigerator.
3. Use wash solution transfer program 1 to transfer 7 mL of high salt wash buffer (pH 6.0) to each filter-cup.
4. Complete liquid filtration through filter cups.
5. Use wash solution transfer program 2 to transfer 3 mL of IX wash buffer (pH 7.4) to each filter-cup.
6. Complete liquid filtration through filter cups.
7. Stop vacuum pump.
8. Use media transfer program to transfer 2.5 mL of Brain Heart Infusion medium to each cup. Is there any mix-
ing step here to dislodge the spores from the filter? The vacuum suction may lead to spore getting stuck to the
filter in the cup.
9. Transfer upper part of manifold to capping tray fitted with bottom caps. Press down to cap bottom of filter-
cups. Bleach bottom part of manifold. Change gloves.
10. When performing standard RV-PCM protocol (16 far incubation):
• Use PCR aliquot program to transfer 60 (iL of sample from each filter cup to a 96-well PCR plate.
• Seal TO sample plate with foil seal. Change gloves.
« Place TO 96-well plate in thermocycler for lysis: 20 minutes at 95°C. Once cycle is completed and plate is
at room temperature, bag 96-well plate in zip-lock bag. bleach the bag.
• Transfer bagged 96-well plate to freezer and store at -20°C until qPCR is run (use a photo-tray to transport
plate).
When performing the optimized RV-PCR protocol (9 hr incubation and Promega DNA extraction and
purification protocol):
• Manually mix sample by swirling pipette tip gently in filter cup, aliquot 120 jjJL volume out of 3 filter cups
(3 replicate samples spiked at a level of 102 or 103 CPU/sample) and transfer 100 joL to 2 mL eppendorf
tubes.
• Add 900 nL of BHI to each tube.
* Add 500 pg of extracted Bg DNA to each tube.
• Process 1 mL sample according to Promega MD1630 protocol described below.
11. Cap top of filter-cups. Double-bag filter-cup manifold. Bleach bag.
12. Transfer bagged filter-cup tray to shaker incubator at 37°C, speed 230 rpm. Incubate for 16 far for standard
RV-PCR protocol or for 9 far for optimized RV-PCR protocol with DNA extraction and purification.
13. Follow laboratory cleanup protocol in Appendix E.
Semi-automated RV-PCR (T16 for standard protocol)
I. After incubation, take filter-cup manifold out of incubator.
2. Vortex filter cup manifold for 10 min on platform vortcxer.
3. Transfer filter-cup manifold to Janus robotic enclosure. Discard bag and uncap filter cups.
4. Use PCR aliquot program to transfer 60 \\L of sample from each filter cup to a 96-well PCR plate (T16
sample plate) according to plate layout. Seal T16 sample plate with foil seal. Change gloves.
5. Lyse plate at 95°C for 20 min on thennocyclcr.
6. Once lysis is completed and plate is at room temperature, bag 96-well plate in zip-lock bag, bleach the bag,
and transfer 96-well crude lysate plate to freezer and store at -20°C until qPCR is run (use a photo-tray to
transport plate).
7. Cap filter cup tray, bag, bleach bag, and transfer to BSC to continue with the Promega DNA extraction and
purification Protocol.
8. Follow laboratory cleanup protocol in Appendix E.
-------
Manual Promega MD1630 DNA Extraction Purification Protocol (T9 for optimized protocol)
1. After incubation, take filter-cup manifold out of incubator.
2. Vortex filter cup manifold for 10 min on platform vortexer.
3. Transfer filter-cup manifold to BSC. Discard bag and uncap filter cups.
DNA extraction and purification phase:
4. Aliquot 1000 jjJL volume out of each filter cup to 2 mL eppendorf tube using a micropipette (T9 samples). Do
not use the 1.5 mL eppendorf tubes.
5. 1st lysis: add 600 joL of bead mix (lysis buffer + magnetic beads) to each tube and mix.
6. Add 360 uL of lysis buffer to each tube. Mix, place on magnet, discard supernatant to waste.
7. 2nd lysis: add 360 ^L of lysis buffer to each tube. Mix, place on magnet, discard supernatant to waste.
8. 1st salt wash: add 360 jjL of salt wash solution to each tube. Mix, place on magnet, discard supernatant to
waste. Repeat one more time.
9. 1st alcohol wash: add 360 jj,L of alcohol wash solution to each tube. Mix. place on magnet, discard
supernatant to waste. Repeat one more time.
10. Air dry (2 min)
11. Heat dry on heal block at 80°C until samples are dry. Allow all alcohol solution to evaporate since alcohol
may interfere with qPCR.
DNA concentration and clution phase:
12. Move sample tubes out of heat block and add 200 \\L of elution buffer to each tube. Mix, place on magnet,
collect supernatant with micropipette and transfer to clean eppendorf tubes (typically, 80 j,iL are collected).
Visually verify absence of magnetic bead cany over during final transfer. If magnetic bead carry over
occurred, place tube back on magnet, collect supernatant, and transfer to clean tube.
13. Store DNA samples at 4 °C until qPCR is run (use photo-tray to transport sample tubes).
14. Follow laboratory cleanup protocol in Appendix E.
PCR for RW-PCR protocol with 16 hr incubation (follow PCR conditions provided in
Appendix F) - Perform on TO T16
1. Set up PCR plate with PCR mix according to plate layout in PCR-preparation hood, seal, transfer to BSC.
2. Take 96-well sample crude lysate plate out of freezer (use a photo-tray to transport, plate), transfer to BSC.
Change gloves. Crude lysate plate should be kept on a cool block or at 4°C at all times.
3. Using BSL3 centrifugation technique (PCR plates are loaded in safety cups), centrifuge sealed sample plate
for 1 min at 2000 rpm.
4. Open crude lysate plate in BSC.
5. Mix samples up and down 10 times and transfer 5 jjJL from lysis plate to the PCR plate (with PCR mix). Seal
PCR plate with clear seal. Change gloves.
When working with dirty wipes, perform 1:10 dilution of crude cell lysate:
Add 90 joL of PCR-grade water to wells of a sterile 96-well bioblock.
Mix crude cell lysate up and down 5 times and transfer 10 |J,L to bioblock wells, maintaining the plate layout.
Mix diluted samples up and down 10 times and transfer 5 \\L from bioblock well to the PCR plate (with PCR
mix). Seal PCR plate with clear seal. Change gloves.
6. Using BSL3 centrifugation technique, centrifuge sealed PCR plate for 1 min at 2000 rpm.
7. Open safety cup in BSC, place plate on photo-tray, change gloves, transfer PCR plate to ABI thermocycler.
-------
8. Run PCR (see Appendix F).
9. After thermal cycling completion, discard sealed PCR plate to waste. Autoclave. PCR plates with amplified
product are never to be opened in the laboratory.
10. Follow laboratory cleanup protocol in Appendix E.
PCR for optimized RV-PCR protocol with 9 hr of incubation Promega DNA extraction
purification (follow PCR conditions provided in Appendix F) - Perform on TO T9
I. Set up PCR plate with PCR mix according to plate layout in PCR-preparation hood, seal, transfer to BSC.
2. If sample tubes were frozen, transfer them to BSC and let them thaw to room temperature.
3. For TO samples: mix samples up and down 10 times and transfer 5 p.L from sample tubes to Hie PCR plate
(with PCR mix). Seal PCR plaie with clear seal. Change gloves.
4. Perform 1:10 dilution of T9 samples:
Add 90 (oL of PCR-grade water to wells of a sterile 96-well bioblock.
Mix sample up and down 5 times and transfer 10 \iL to bioblock wells, maintaining the plate layout.
Mix diluted samples up and down 10 times and transfer 5 \,iL from bioblock well to the PCR plate (with PCR
mix). Seal PCR plate with clear seal. Change gloves.
5. Using BSL3 centrifugation technique, centrifuge sealed PCR plate for 1 min at 2000 rpm.
6. Open safety cup in BSC, place plate on photo-tray, change gloves, transfer PCR plate to AB1 thermocycler.
7. Run PCR (Please see Appendix F).
8. After thermal cycling completion, discard sealed PCR plate to waste. Autoclave. PCR plates with amplified
product are never to be opened in the laboratory.
9. Follow laboratory cleanup protocol in Appendix E.
-------
-------
Appendix C:
Final Manual RV-PCR Protocol
Qualitative of in wipe, air filter
Laboratory set-up
* Put PPE (personal protective equipment) on: lab coat, safer}' glasses, double gloves.
• Prepare fresh bleach solution (1 volume bleach + 9 volumes water). Date and label with initials.
* Clean/bleach BioSafety Cabinet (BSC) and bench surfaces.
* All sample manipulations are performed in the BSC
Place in
* Wipes and air filters: Using sterile forceps, transfer sample collection type to 30 mL tube behind internal mesh/
support (1.5 mL of IX wash buffer is added to each wipe prior to spiking).
• Water samples: No mesh support is needed. Aliquot 20 mL of water directly in 30 mL tube.
Plating
Although no plating is included in the RV-PCR method, it is suggested to preserve samples for any further
confirmation using plating and other microbiological culture-based methods.
Preparation of BioSafety Cabinet for manual RV-PCR
Materials:
• Bleach wipes and daily bleach bottle
« Sharps waste container
* Absorbent pad
• Beaker with top filter-cup caps
• Capping tray with bottom caps
• 2 large biohazard bags + rubber bands
« Allen wrench for manifold
* Serological pipettes and pipettor
Set up:
« Set up manifold: connect vacuum pump, pump in line filter, and waste container with fresh bleach; tape pump
exhaust tube to BSC to vent exhaust inside BSC
• Add filter cups to manifold
• Tape filter-cup layout on glass window of the BSC
-------
Preparation of the cart for manual RV-PCR
Materials:
• Gloves
• Bleach wipes
« Waste bags
* Absorbent pads
• Isopropyl alcohol squeeze bottle
• Deionized water squeeze bottle
• Red biohazards bags + rubber bands
« Autoclave tape
* Large photo-tray
• Sharps container
* 2 niLeppendorf tubes
• 200 p,L pipettor and pipette tips
* Marker
Media and Buffers:
* Brain Heart Infusion medium
• High salt wash buffer (pH 6.0)
« IX wash buffer (pH 7.4)
Manual protocol (TO)
1. In the BSC, add 20 mL of extraction buffer to wipe or air filter samples placed in a 30 mL tubes (no addition
is needed for 20 mL water samples).
2. Seal sample rack in waste bag; bleach exterior of bag with bleach wipe.
3. Vortex samples for 20 min on platform vortexer (outside BSC), position 7.
4. After vortexing samples, transfer sample rack to BSC. Remove tube rack from plastic bag.
5. Turn on vacuum pump at 10 psi.
6. Using BSL3 pipetting technique, transfer sample from 30 mL sample tube to filter-cup using a serological
pipette (the total sample volume is 20 mL-. Pipette 15 mL and deliver 13 mL- to filter-cup). Dispose pipette
in waste container. Cap sample tube, bleach the outer surface of sample tube. Re-place tube back on rack.
Change gloves.
7. Repeat step 6 for each sample.
8. Complete filtration of liquid through filter cups.
9. Bleach original sample rack. Double-bag rack. Bleach outer surface of biohazard bag. Change gloves.
10. Transfer double-bagged original sample rack to refrigerator.
11. Transfer 7 mL of high salt wash buffer (pH 6.0) to each filter-cup using a serological pipette. Use new
disposable pipette for each sample cup.
12. Complete filtration of liquid through filter cups.
13. Transfer 3 mL of IX wash buffer (pH 7.4) to each filter-cup using a serological pipette. Use new disposable
pipette for each sample cup.
14. Complete filtration of liquid through filter cups. Change gloves.
15. Stop vacuum pump.
16. Bring 5 mL graduated pipettes, 200 |j.L pipettor, 200 |j.L tips, BHI medium, sharps container, centrifuge safety
cup, zip-lock bag with PCR plate, plate support, foil seals, and plate sealer to BSC. Set up work space in
BSC. Change gloves.
17. Pipette 2.5 mL of Brain Heart Infusion medium into each filter cup using a serological pipette. Use new
disposable pipette for each filter cup.
-------
18. Transfer upper part of manifold to capping tray fitted with bottom caps. Press down to cap bottom of filter-
cups. Change gloves.
19. For each sample: swirl pipette tip gently in filter cup to mix sample, aliquot 120 (oL volume out of each cup
using a micropipette. and transfer 100 p.L to 2 rriL eppendorf tubes.
Add 900 j,iL of BH1 to each eppendorf tube.
Add 500 pg of extracted Bg DNA to each eppendorf tube.
Process 1 niL samples according to Promega MD1630 protocol described below.
20. Cap filter-cups. Double-bag filter-cup manifold. Bleach bag.
21. Transfer bagged filter-cup tray to shaker incubator at 37°C, speed 230 rpm. Incubate for 9 hr.
22. Follow laboratory cleanup protocol in Appendix E.
Manual Promega MD1830 DNA Extraction Purification Protocol (T9)
I. After incubation, take filter-cup manifold out of incubator.
2. Vortex filter cups for 10 min on platform vortexer.
3. Transfer filter-cup manifold to BSC.
DNA extraction and purification phase:
4. Aliquot 1000 (iL volume out of each filter cup to 2 rnL eppendorf tube using a micropipette (T9 samples). Do
not use the 1.5 mL eppendorf tubes.
5. 1st lysis: add 600 jjJL of bead mix (lysis buffer + magnetic beads) to each TO and T9 tubes and mix.
6. Add 360 ^L of lysis buffer to each tube. Mix, place on magnet, discard supernatant to waste.
7. 2nd lysis: add 360 jjL of lysis buffer to each tube. Mix. place on magnet, discard supernatant to waste.
8. 1st salt wash: add 360 p,L of salt wash solution to each tube. Mix. place on magnet, discard supernatant to
waste. Repeat one more time.
9. lsl alcohol wash: add 360 |_iL of alcohol wash solution to each tube. Mix, place on magnet, discard
supernatant to waste. Repeat one more time.
10. Air dry (2 min)
11. Heat dry on heat block at 80°C until samples are dry. Allow all alcohol solution to evaporate since alcohol
may interfere with qPCR.
DNA concentration and elution phase:
12. Move sample tubes out of heat block and add 200 (oL of elution buffer to each tube. Mix, place on magnet,
collect supernatant with micropipette and transfer to clean eppendorf tubes (typically, 80 p.L are collected).
Visually verify' absence of magnetic bead cany over during final transfer. If magnetic bead carry over
occurred, place tube back on magnet, collect supernatant, and transfer to clean tube.
13. Store DNA samples at 4 °C until qPCR is run (use photo-tray to transport sample tubes).
14. Follow laboratory cleanup protocol in Appendix E.
-------
PCR (follow PCR conditions provided in Appendix F) - Perform on TO T9 samples
1. Set up PCR plate with PCR mix according to plate layout in PCR-preparation hood, seal and transfer to BSC.
2. Transfer sample tubes to BSC.
3. For TO samples: mix samples up and down 10 times and transfer 5 j,iL from sample tubes to the PCR plate
(with PCR mix). Seal PCR plate with clear seal. Change gloves.
4. Perform 1:10 dilution of T9 samples:
Add 90 |j,L of PCR-grade water to wells of a sterile 96-well bioblock.
Mix sample up and down 5 times and transfer 10 j,iL to bioblock wells, maintaining the plate layout.
Mix diluted samples up and down 10 times and transfer 5 ^L from bioblock well to the PCR plate (with PCR
mix). Seal PCR plate with clear seal. Change gloves.
5. Using BSL3 centrifugation technique, centrifuge sealed PCR plate for 1 min at 2000 rpm.
6. Open safety cup in BSC, place plate on photo-tray, change gloves, transfer PCR plate to AB1 thermocycler.
7. Run PCR (Please see Appendix F).
8. After thermal cycling completion, discard sealed PCR plate to waste. Autoclave. PCR plates with amplified
product are never to be opened in the laboratory.
9. Follow laboratory' cleanup protocol in Appendix E.
-------
D:
Qualitative of in wipe, air filter
Laboratory set-up
* Put PPE (personal protective equipment) on: lab coat, safer}' glasses, double gloves.
• Prepare fresh bleach solution (1 volume bleach + 9 volumes water). Date and label with initials.
* Clean/bleach BioSafety Cabinet (BSC), Janus enclosure and bench surfaces.
* All sample manipulations are performed in the BSC or in the HEPA-filtered Janus enclosure.
Place in
* Wipes and air filters: Using sterile forceps, transfer sample collection type to 30 mL tube behind internal mesh/
support (1.5 mL of IX wash buffer is added to each wipe prior to spiking).
• Water samples: No mesh support is needed. Aliquot 20 mL of water directly in 30 mL tube.
Plating
Although no plating is included in the RV-PCR method, it is suggested to preserve samples for any further
confirmation using plating and other microbiological culture-based methods.
Preparation of the Janus Robotic enclosure
Materials:
• Bleach wipes and daily bleach bottle
« Sharps waste container to collect tips
* Beaker with top filter-cup caps
• Capping tray with bottom caps
• 2 large biohazard bags + rubber bands
• Allen wrench for manifold
Setup:
• Set up manifold: connect vacuum pump, pump in line filter, and waste container with fresh bleach; tape pump
exhaust tube to Janus enclosure to vent exhaust inside enclosure
« Add filter cups to manifold
* Tape filter-cup layout on glass window of the enclosure
• Tape PCR plate layout on glass window of the enclosure
-------
Preparation of the cart
Materials:
• Gloves
• Bleach wipes
« Waste bags
* Isopropyl alcohol squeeze bottle
• Deionizcd water squeeze bottle
• Red biohazards bags + rubber bands
• Autoclave tape
« Large photo-tray
* Reservoirs
• Sharps containers
* Robotic tip refills
• Zip-lock bag containing: 96-well plate, plate support, foil seals, and plate sealer
• 2 mLeppendorf tubes
* Pipettor and pipette tips: 200 jaL
« Marker
Media and Buffers:
« Brain Heart Infusion medium
« High salt wash buffer (pH 6.0)
• IX wash buffer (pH 7.4)
preparation in BSC
I. Using BSL3 pipetting technique, add 20 mL of extraction buffer to wipes and air filter samples placed in a 30
mL tubes (not needed for 20 mL water samples).
2. Seal sample rack in plastic bag; bleach bag.
3. Vortex samples for 20 min on platform vortexer, position 7 (outside BSC).
4. After vortexing samples, transfer sample rack to Robotic enclosure. Remove tube rack from plastic bag.
RV-PCR (TO)
1. Turn on vacuum pump at 10 psi.
2. Using the Janus robotic system, transfer 13 mL from 30 mL sample tubes to filter-cups. Stop robot, cap
sample tubes, bleach the outer surface of sample tubes, bag the original sample tube rack, bleach the bag, and
transfer to refrigerator.
3. Use wash solution transfer program 1 to transfer 7 mL of high salt wash buffer (pH 6.0) to each filter-cup.
4. Complete liquid filtration through filter cups.
5. Use wash solution transfer program 2 to transfer 3 mL of IX wash buffer (pH 7.4) to each filter-cup.
6. Complete liquid filtration through filter cups.
7. Stop vacuum pump.
8. Use media transfer program to transfer 2.5 mL of Brain Heart Infusion medium to each cup. Is there any
mixing step here to dislodge the spores from the filter? The vacuum suction may lead to spore getting stuck to
the filter in the cup.
9. Transfer upper part of manifold to capping tray fitted with bottom caps. Press down to cap bottom of filter-
cups. Bleach bottom part of manifold. Change gloves.
-------
10. For each sample: Use PCR aliquot program to transfer 100 |j,L volume out of each filter cup and transfer 100
joL to 2 mL eppendorf tubes.
Add 900 nL of BHI to each tube.
Add 500 pg of extracted Bg DNA to each tube.
Process 1 mL sample according to Promega MD1630 protocol described below
11. Cap top of filter-cups. Double-bag filter-cup manifold. Bleach bag.
12. Transfer bagged filter-cup tray to shaker incubator at 37°C, speed 230 rpm. Incubate for 9 hr.
13. Follow laboratory cleanup protocol in Appendix E.
Manual Promega MD1630 DNA Extraction Purification Protocol (T9)
I. After incubation, take filter-cup manifold out of incubator.
2. Vortex filter cup manifold for 10 min on platform vortcxcr.
3. Transfer filter-cup manifold to BSC. Discard bag and uncap filter cups.
DNA extraction and purification phase:
4. Aliquot 1000 (oL volume out of each filter cup to 2 mL eppendorf tube using a micropipette (T9 samples). Do
not use the 1.5 mL eppendorf tubes.
5. 1st lysis: add 600 jjL of bead mix (lysis buffer + magnetic beads) to each TO and T9 tubes and mix.
6. Add 360 j,iL of lysis buffer to each tube. Mix, place on magnet, discard supernatant to waste.
7. 2"d lysis: add 360 (oL of lysis buffer to each tube. Mix, place on magnet, discard supernatant to waste.
8. 1st salt wash: add 360 jaL of salt wash solution to each tube. Mix. place on magnet, discard supernatant to
waste. Repeat one more time.
9. 1st alcohol wash: add 360 joL of alcohol wash solution to each tube. Mix, place on magnet, discard
supernatant to waste. Repeat one more time.
10. Air dry (2 min)
11. Heat dry on heat block at 80°C until samples are dry. Allow all alcohol solution to evaporate since alcohol
may interfere with qPCR.
DNA concentration and elution phase:
12. Move sample tubes out of heat block and add 200 (oL of elution buffer to each tube. Mix, place on magnet,
collect supernatant with micropipette and transfer to clean eppendorf tubes (typically, 80 (oL are collected).
Visually verify absence of magnetic bead carry over during final transfer. If magnetic bead cany over
occurred, place tube back on magnet, collect supernatant, and transfer to clean tube.
13. Store DNA samples at 4°C until qPCR is run (use photo-tray to transport sample tubes).
14. Follow laboratory cleanup protocol in Appendix E.
-------
PCR (follow PCR conditions provided in Appendix F) - Perform on TO T9 samples
1. Set up PCR plate with PCR mix according to plate layout in PCR-preparation hood, seal and transfer to BSC.
2. Transfer sample tubes to BSC and let them thaw to room temperature.
3. For TO samples: mix samples up and down 10 times and transfer 5 joL from sample tubes to the PCR plate
(with PCR mix). Seal PCR plate with clear seal. Change gloves.
4. Perform 1:10 dilution of T9 samples:
Add 90 |j,L of PCR-grade water to wells of a sterile 96-well bioblock.
Mix sample up and down 5 times and transfer 10 j,iL to bioblock wells, maintaining the plate layout.
Mix diluted samples up and down 10 times and transfer 5 ^L from bioblock well to the PCR plate (with PCR
mix). Seal PCR plate with clear seal. Change gloves.
5. Using BSL3 centrifugation technique, centrifuge sealed PCR plate for I min at 2000 rpm.
6. Open safety cup in BSC, place plate on photo-tray, change gloves, transfer PCR plate to AB1 thermocycler.
7. Run PCR (Please see Appendix F).
8. After thermal cycling completion, discard sealed PCR plate to waste. Autoclave. PCR plates with amplified
product are never to be opened in the laboratory.
9. Follow laboratory cleanup protocol in Appendix E.
-------
E:
Lab and
Cleanup Protocol
* Dispose of all biological materials in autoclave bags (double bagged)
* Autoclave all waste materials
* Decontaminate counters and all equipment with fresh bleach (1 volume water and 9 volumes commercial bleach),
followed by 70% isopropanol, and finally rinse with DI water.
1 M Sodium Hydroxide Solution (NaOH)
Note: Label all bottles and flasks with reagent name, dale and initials.
1. Weigh 10 g NaOH
2. Transfer 10 g NaOH to 200 mL MilliQ H2O
3. Mix with magnetic stirrer
4. After NaOH pellets are dissolved, bring final volume to 250 mL with MilliQ H2O
10X (250 mM KH2PO4, pH 7.4)
Note: Label all bottles and flasks with reagent name, date and initials.
1. Dissolve 34 g KH2PO4 in 500 mL MiliQ H,O
2. Add enough 1 M NaOH to bring to pH 7.4 (> 200 mL of 1 M NaOH)
3. Bring to volume with MilliQ H2O to 1 L
4. Filter sterilize using 1 L, 0.22 micron cellulose acetate filtering system with disposable bottle
1 X (25 mM KH2PO4, pH 7.4)
Note: Label all bottles and flasks with reagent name, pH level, date and initials.
1.50 mL 1 OX wash buffer
2. 950 mL MilliQ H,O
3. Mix with magnetic stirrer, when mixed, measure pH
4. Filter sterilize using 1 L. 0.22 cellulose acetate filtering system with disposable bottle
High Salt-Low pH (207 mM KHZPO4, pH 6.0)
Note: Label all bottles and flasks with reagent name, dale and initials.
Dissolve 28.2 g KH,PO4 in 500 mL MiliQ H2O
1. Add enough 1 M NaOH to bring to pH 6.0 (> 100 mL of 1 M NaOH)
2. Bring volume with MilliQ H2O to 1 L
3. Filter sterilize using 1 L, 0.22 micron cellulose acetate filtering system with disposable bottle
-------
Extraction mMKH2POf 30 % 0.1% Tween 80)
1. 698niLMiiiiQH,O
2. 1 inL 10X wash buffer
3. 300 mL 200 proof ctlianol
4. ImL Tween* 80
5. Filter sterilize using 1 L. 0.22 cellulose acetate filtering system with disposable bottle
Brain Heart Infusion Agar
1. 26 g Bacto Brain Heart Infusion agar powder
500 mL MilliQ H9O
2. Place on hotplate and gently mix with spin bar.
3. Autoclave
4. Place on hotplate and gently mix with spin bar. Allow agar to cool down to 45°C before pouring.
5. Pour 20 mL of solution in each petri dish using a serological pipette. Pouring is performed in a sterile BSC.
-------
Appendix F:
PCR conditions
Reagents:
Primers and probes
TaqMan 2X Universal Master Mix with UNO and AmpliTaq Gold (ABI cat. Number 4305719)
Molecular Biology grade distilled water, RNase- and DNase-free (Teknova cat. Number W3350)
PCR mix for pXO2 (EPA-2) primer/probe set
Reagent
TaqMan 2X Universal Master Mix
Forward primer, 25 uM
Reverse primer, 25 uM
Probe, 2 uM
Molecular Biology Grade Water
Template DNA
TOTAL
Volume (uL)
12.5
0.3
0.3
1
5.9
5
25
Final Concentration (jiM)
IX
0.3
0.3
0.08
N/A
Variable
PCR mix for Chromosome (BC3) and pXOl (EPA-1) primer/probe sets.
Reagent
TaqMan 2X Universal Master Mix
Forward primer, 25 uM
Reverse primer, 25 uM
Probe, 2 uM
Molecular Biology Grade Water
Template DNA
TOTAL
Volume (uL)
12.5
1
1
1
4.5
5
25
Final Concentration (uM)
IX
1.0
1.0
0.08
N/A
Variable
Equipment:
ABI 7500 fast thermocycler
Optical Fast 96-well plates (ABI, cat. Number 4366932)
Optical adhesive plate covers (ABI, cat. number 4311971)
PCR conditions for all three primer/probe sets:
STEPS
Temperature
Time
UNG incubation
HOLD
50°C
2 min
AmpliTaq Gold
activation
HOLD
95°C
10 min
PCR , 45 cycles
Denaturation
95°C
5 sec
Annealing/extension
60°C
20 sec
*Fast Ramp: 3.5 oC/sec up and 3.5oC/sec down.
-------
-------
Appendix G:
Consumables
Description
Catalog #
Units
Supplier
Item part Number
PCR Materials
PCR plates
PCR plates seals
Universal PCR master mix
PCR probes
PCR primers
96 Well Hard Plates Costar-black
100/cs
2.0 mL Screw cap tubes (500 bag)
4346906
4311971
4305719
custom
MD1360
29442-922
20170-237
20/box
100/box
(10) 5 mL bottles
96 reactions
cs
bag
Applied Biosystems,
INC.
Applied Biosystems,
INC.
Applied Biosystems Inc
(ABI)
Biosearch Technologies
Biosearch Technologies
GSS
GSS
4366932
4311971
4305719
DLO-FB1-1
P2C-1
29442-922
20170-237
Culture Materials
BHI media
BHI agar
TSB Agar
TSB Media
Petri dishes 60x1 5
LAZY-L-SPREADERS STERILE
case of 500
237500
241830
25373-085
101100-886
500 g / bottle
500 g /bottle
cs
500/cs
VWR
VWR
VWR
VWR
GSS
Government Scientific
source (GSS)
90004-690
90003-262
200059-616
90004-270
25373-085
101100-886
Buffers
Potassium phosphate, KH2PO4
Tween® 80 9005-65-6
EtOH 200proof absolute,
anhydrous
Distilled Water
22, 130-9
103170
111ACS200
W3350
500 g / bottle
100 mL
1L
1L
Aldrich
MP Biomedicals
TRANS MERIDIAN
UCI/QUANTIUM
CHEM
Quality Biological Inc.
221309
103170
351-029-721
DNA extraction and purification reagents
Promega Kit
Promega salt wash
Promega beads
Promega lysis buffer
Promega anti-foam
Promega
Promega
Promega
Promega
Promega
MD1360
MD1401
MD1441
MD1392
MD1431
-------
Vacuum filtration materials
Tygon Tubing 1/4" ID 1/2" OD
Nalgene 2 Liter Bottle
Nalgene Venting Cap
3870E DOOR BELLOWS
ASSEMBLY KIT - Part for
vacuum pump
3870E AIR JET VALVE (BLACK
TOP) - Part for vacuum pump
3870E DOOR GASKET (Door
Seal) - Part for vacuum pump
3870E FILL/VENT MESH
CHAMBER FILTER (Stainless
Steel) - Part for vacuum pump
3870E PLUNGER VALVE KIT
(3mm) - Part for vacuum pump
3870E PLUNGER VALVE KIT
(6mm) - Part for vacuum pump
3870E SAFETY VALVE (40 PSI)
- Part for vacuum pump
BH-95636-00
BH06257-20
BH06258-10
TUK030-2150
TUJ034-2149
TUG074-2146
MIF062-2126
TUK082-2155
TUK086-2156
TUV065-2166
bx
ea
ea
ea
ea
ea
ea
ea
ea
ea
Cole-Parmer
Cole-Parmer
Cole-Parmer
A & A Dental &
Medical Services
A & A Dental &
Medical Services
A&ADental&
Medical Services
A & A Dental &
Medical Services
A & A Dental &
Medical Services
A&ADental&
Medical Services
A&ADental&
Medical Services
BH-95636-00
BH06257-20
BH06258-10
TUK030-2150
TUJ034-2149
TUG074-2146
MIF062-2126
TUK082-2155
TUK086-2156
TUV065-2166
Sample processing materials
30 mL Screw blue cap tube
Polyethylene caps
Monofilament polyester mesh disc,
Quick turn tube fitting
polypropylene, female cap
Whatman autocups [available from
VWR under misc-supplies]
Disposable Nylon Forceps
100 mL Reagent Reservoirs
(100/case)
Bioblocks for dilutions ( 96 wells/
2mLper well)
EK-T3242S
94075K56
93185 T17
51525K365
1602-0465
12576-933
8086
662000
100/cs
pk
ea
pk
100/pack
cs
20/case
E & K Scientific
McMaster Carr
McMaster Carr
Ark-Plas Products, Inc.
VWR
Government Scientific
source (GSS)
Thermo Fisher
E & K Scientific
T3242S
57935K16
3185T17
AP17FLPOOP
1502-0465(Whatman)
12576-933
8086
662000
Robotic materials
Robotic tips
Large reservoirs for robot
Sharps container for Janus
6001256
2035
33000-956
10 racks/ case
25/case
32/case
Perkin Elmer
E & K Scientific
GSS
6001256
EK-2035
33000-956
Pipettors and tips for PCR and DNA extraction and purification
1 000 uL Filter LTS Tips
200 uL Filter LTS Tips
20 uL Filter LTS Tips
L- 1000 LTS Pipettor
L-lOOLTSPipettor
L-200 LTS Pipettor
L-20 LTS Pipettor
L- 10 LTS Pipettor
Carousel Stand
RT-L10F
RT-L1000F
RT-L200F
L1000 LTS
L100 LTS
L200 LTS
L20 LTS
L10 LTS
CR-7
cs
cs
cs
ea
ea
ea
ea
ea
ea
Rainin
Rainin
Rainin
Rainin
Rainin
Rainin
Rainin
Rainin
Rainin
RT-L20F
RT-L1000F
RT-L200F
L1000 LTS
L100 LTS
L200 LTS
L20 LTS
L10 LTS
CR-7
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General laboratory supplies
Diamond Grip Latex Gloves
X-Small
Diamond Grip Latex Gloves Small
Diamond Grip Latex Gloves
Medium
Diamond Grip Latex Gloves Large
Diamond Grip Latex Gloves
X-Large
VWR Autoclave Bags 25x35
VWR 5.0 mL Freezer Vials
Corning 50 mL Conical centrifuge
tubes
Corning 15 mL Conical centrifuge
tubes
BD Sharps Containers
Kaydry EX-L Wipers
Bleach gallon bottles case
Sleeves protectors
Disposables lab. coats w/cuffs
VWR, Bleach wipes 10 pkg/cs
Heavy Duty Waste Bags
Autoclave ampoules
Wipers
32916-498
32916-506
32916-500
32916-502
32916-503
14220-042
66008-400
21008-714
21008-678
BD305551
21903-021
37001-060
10832-668
CV9841N
47735-634
436188D4
101101-788
cs
cs
cs
cs
cs
cs
bag
cs
cs
cs
cs
cs
cs
cs
lOpkg/cs
20 ampules/ box
GSS
GSS
GSS
GSS
GSS
GSS
GSS
GSS
GSS
GSS
GSS
GSS
ValuMax International
GSS
Government Scientific
source (GSS)
Staples
Government Scientific
source (GSS)
Government Scientific
source (GSS)
32916-498
32916-506
32916-500
32916-502
32916-503
14220-032
66008-400
89004-367
89004-370
BD305551
21903-021
37001-060
1919W
CV9841N
37001-060
Item 436188
Model WHD3339
14220-030
37002-030
Serological pipettor and tip for manual RV-PCR only
Portable pipet aid
50 mL serological pipettor tips
10 mL serological pipettor tips
25 mL serological pipettor tips
5 mL serological pipettor tips
4-000-100
29442-440
29442-430
29442-436
29442-422
bag
bag
bag
bag
VWR
GSS
GSS
GSS
GSS
4-000-100
53283-712
53283-708
53283-710
53283-706
Sample materials
Aerosol niters
Wipes
FSLW04700
Kendall #8042
100 filters/ box
3000/case
Millipore
GSS
FSLW04700
89004-507
Acronyms: bx, box; cs, case; ea, each; pk, package
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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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