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| Validation of PCR Assays for the Detection of Bacillus anthracis Spores The Plan AOAC is working closely with method volunteers and scientists from the Critical Reagents Program (CRP) and National Guard Bureau (NGB) to develop a series of validation test plans to evaluate three assay systems: Department of Defense (DOD) ABI, DOD Joint Biological Agent Identification and Diagnostic System (JBAIDS), and NGB JBAIDS. The validation test plans have been carefully constructed to compliment ongoing projects sponsored by the Department of Homeland Security (DHS). This unique joint evaluation using the identical test samples, collaborating laboratories, laboratory personnel, and test plans will allow for complete comparability of the systems. Edgewood Chemical Biological Center (ECBC) is serving as the lead laboratory, with U.S. Army Dugway Proving Ground as the sample preparation laboratory. Both are participating as collaborators. In addition, 10 other collaborating laboratories have been recruited, trained, and tested in a trial run that was performed in January 2008. The validation will be completed by the end of June 2008. The Players CRP, a program office located near the ECBC, falls under the Joint Program Executive Office for Chemical Biological Defense, within the Chemical Biological Medical Systems Joint Project Management Office. CRP’s mission is to develop, produce, and store the highest quality detection assays and reagents as a national resource for DOD and the Biological Defense Community. DOD and other agencies, including the Department of Health and Human Services (DHHS) and DHS, use CRP reagents and assays. The CRP is actively promoting joint programs with other federal agencies, such as the NGB, to create a nationally accepted process for standardizing reagents, tests, and methods. This program will increase cross-agency confidence to provide reliable information, and will enable the acceptance of new assays and platforms in a timely and quality controlled manner. NGB has also newly deployed a system to monitor biological threat agents known as JBAIDS, a diagnostic test platform used by several of the military services to confirm the presence of biological warfare agents and toxins. NGB has joined with CRP in this unique, joint validation effort. Serving as the lead laboratory, ECBC is a world leader in applying state-of-the-art science, technology, and engineering to chemical and biological defense problems. Application of chemical and biological technologies developed at ECBC has protected U.S. soldiers for the last 85 years during every major war or conflict in which the United States was involved. ECBC is located at the Edgewood Area of Aberdeen Proving Ground. The Assays CRP and NGB use assays based on polymerase chain reaction (PCR), a common technique widely used in medical and food microbiology methods. The name of the technique is derived from one of its key components, a DNA polymerase used to amplify a piece of DNA by enzymatic replication. As the PCR reaction progresses, the newly replicated DNA is used as a template for further replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR, it is possible to amplify a few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. Such PCR assays can be very selective and sensitive when primers are highly complementary to the DNA region targeted for amplification. The CRP assay is based on the ABI 7900HT Standard Block platform manufactured by Applied Biosystems and uses three unique primer pairs and probes. The NGB assay uses JBAIDS, a unique platform developed for military use and intended to provide rapid identification and diagnostic confirmation. The JBAIDS is designed as a reusable, portable, and modifiable system capable of simultaneous, reliable identification of multiple biological warfare agents and pathogens of operational and clinical significance. The system is man-portable and contains a field-hardened air thermocycler capable of automatically analyzing prepared samples for the presence of targeted DNA sequences. The instrument combines rapid thermocycling technology and a real-time fluorimeter to identify test samples. JBAIDS units can be used with dry filters used in aerosol collection units for air monitoring, or can be used with swabs for surface samples. The CRP assay using the ABI platform will be validated for use with dry filter unit filters. The CRP assay is also being validated with the JBAIDS platform. --Scott Coates
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