In the event of a biothreat agent release, hundreds to thousands of samples will need to be rapidly processed in order to both characterize the extent of contamination and determine the efficacy of remediation activities. Current viability methods are both labor- and time-intensive such that they cannot meet the need for rapid analysis. In order to close this technical gap, LLNL developed high throughput sample processing protocols integrated with real-time Polymerase Chain Reaction (PCR), expanding its capabilities by conducting PCR analysis pre- and post-incubation and using the change in cycle threshold to selectively detect viable organisms. The approach referred to as Rapid Viability (RV)-PCR allows detection of low levels of viable organisms in the presence of environmental backgrounds and high populations of live non-target microorganisms or dead target spores. Results obtained with virulent Bacillus anthracis in water, wipes and air filters with both manual and automated methods will be presented. Multiple assays targeting both the chromosome and plasmids were used to increase confidence in specific pathogen detection. Criteria for RV-PCR protocols included limits of detection, accuracy with plating and turn-around time for results. This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. Funding for this research was provided by the National Homeland Security Research Center of the U.S. Environmental Protection Agency. LLNL-ABS-416418.
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