oEPA
technical BR
Rapid Viability PCR Method for Detection of Live Bacillus anthracis
Spores
Introduction
To protect human health and ensure that the environment and
facilities are remediated 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
environmental samples of diverse types (aerosol, surface,
water, etc.) could need to be processed. Decision-makers
need rapid results to determine whether facilities and areas
can be re-opened, or if additional decontamination is needed.
Current viability test methods are too labor-, space-, and time-
intensive to be able to meet the need for rapid analysis. With
current methods, only 30-40 samples may be processed each
day per laboratory with confirmation not available until days
later.
EPA's Homeland Security Research Program
(HSRP) develops products based on
scientific research and technology
evaluations. Our products and expertise are
widely used in preventing, preparing for, and
recovering from public health and
environmental emergencies that arise from
terrorist attacks. Our research and products
address biological, radiological, or chemical
contaminants that could affect indoor areas,
outdoor areas, or water infrastructure. HSRP
provides these products, technical
assistance, and expertise to support EPA's
roles and responsibilities under the National
Response Framework, statutory
requirements, and Homeland Security
Presidential Directives.
The EPA's Office of Emergency Management within the Office
of Solid, Waste, and Emergency Response has established the Environmental Response Laboratory
Network (ERLN), a network of laboratories to analyze environmental samples following biological,
chemical, or radiological attacks. Validated, rapid viability test protocols are needed as part of the ERLN
capabilities. 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.
Rapid Viability PCR (RV-PCR)
The EPA-NHSRC developed Rapid Viability PCR (RV-PCR) method determines the presence or
absence of live B. anthracis spores, which is a key analytical requirement during the cleanup phase of a
response. This method can be more sensitive than the traditional culture-based method because RV-
PCR uses the whole sample for analysis. RV-PCR is relatively rapid and cost-effective. It is also less
labor-intensive, less prone to inhibition by environmental matrices, and less prone to interference from
other biological material in the sample than the culture method. It also provides higher-throughput and
generates significantly less bio-hazard and other laboratory wastes than the culture-based method.
The RV-PCR method combines broth culture and real-time PCR, a highly sensitive and specific analytical
method to identify bioagents. The broth culture part of the method allows the germination of B. anthracis
spores recovered from a sample. The real-time PCR part of the method offers a rapid determination of
the identity of B. anthracis bacteria.
The first step of the protocol involves multiple extraction and wash steps through which spores are
recovered in filter cups. Next, a growth media is added to the spores in filter cup for optimum growth of B.
anthracis. An aliquot is withdrawn before incubating the broth culture in the filter cup. This is the Time
Zero (TO) aliquot. After 9 hours of incubation another aliquot is withdrawn. This is the T9 aliquot. The
DNA extracts of the TO and T9 DNA aliquots, are then analyzed via real-time PCR to detect the presence
of B. anthracis. The PCR cycle-threshold (Ct) number for both the TO and T9 aliquots are recorded and
compared. A change (decrease) in the Ct number for the T9 aliquot relative to the Ct number for the TO
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aliquot is calculated. A delta Ct > 9 with an endpoint PCR Ct of <36 for the T9 aliquot, represents an
increase in B. anthracis concentration at T9 relative to TO as a result of viable spores in the sample that
germinated and grew during the 9 hr of incubation for growth. Depending upon the end user requirement
and the phase of response during an event, a lower delta Ct(> 6, to represent at least a two log difference
in DNA concentration), and a corresponding higher end point (PCR Ct of <_39) could be set. It should be
noted that the current version of the method provides qualitative (presence/absence) detection. However,
introduction of the Most Probable Number (MPN) steps in the method could provide some quantitative
analysis.
•mr
1 The RV-PCR method is
based on a shift in PCR Ct
value indicating an increase
in DNA due to growth of
viable organisms
1 The method accurately
distinguishes live cells from
dead spores/cells based on
Ct0, Ctfina| and ACt
1 ACt = 9 for a positive result
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PCR cycle number
The method has significant advantages over the traditional plate method:
• Higher sensitivity (> 10x improvement over plate method)
• No interference with live environmental background (selective method)
• May be fully automated (high-throughput)
• Built-in PCR confirmation
. Faster turn-around time for results
Limit of Detection with RV-PCR
•'.0
Wipes, 10 Spore Level Detection
Water, 10 Spore Level Detection
Filters, 10 Spore Level Detection
• Levels down to 10 spores per sample
were detected after 9 hrs of incubation with
and without debris.
• EPA1 = pXO1 Plasmid PCR Assay
» EPA2 = pXO2 Plasmid PCR Assay
• BC3 = Genomic Target PCR Assay
• Dirty = Spiked with AZ Test Dust
" The total manual processing time from
start to finish for 16 samples was 15 hrs.
January, 2012
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Comparison of RV-PCR with Traditional Microbiological Culture (Plating) Method
PCP& other
Plating
method
Serial dilutions
andplating
Colony counts
obtained-
presumptive
detection
confirmatory methods
Confirmation required
20-40 samples
analyzed
Hours 0
12
18
24
30
36
42
48
DAW
detection & •
RV-PCR
method
Sample analysis cycles continuing
(25 sample data every 3 hours)
~150
samples
analyzed
Results of 1st batch
The RV-PCR method is being transitioned to the OEM (ERLN) and the capability has been established
at the OPP Microbiology Lab at Ft. Meade, MD. Also, the Lawrence Livermore National Laboratory at
Livermore, CA, who developed the RV-PCR method under the lAwith NHSRC, is also an ERLN
member lab to perform RV-PCR based sample analyses. Additionally, the RV-PCR capability will be
established at a laboratory of University of Cincinnati, Cincinnati, OH, and the NEIC at Denver, CO,
The development of RV-PCR methods for Yersinia pestis and Francisella tularensis is also in progress.
Contacts
Dr. SanjivShah (shah.saniiv@epa.gov)
Dr. Tonya Nichols (nichols.tonva@epa.gov)
January, 2012
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