Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2090670 | Journal of Microbiological Methods | 2009 | 7 Pages |
Abstract
To rapidly remediate facilities after a biothreat agent release, improved turnaround times are needed for sample analysis. Current methods to confirm the presence of a viable biothreat agent are limited by low sample throughput. We have developed a rapid-viability-polymerase chain reaction (RV-PCR) method to determine the presence of viable spores. The method combines high-throughput sample processing with 96-well PCR analysis, which measures a change in real-time, quantitative PCR response arising from increased target-cell populations during culturing. The method accurately detects 1 to 10 live spores in a high-dead spore background (106). Field tests using approximately 1000 biological indicators, each containing 106 spores of the B. anthracis surrogate, Bacillus atrophaeus, exposed to seven lethal and sub-lethal chlorine dioxide levels showed no significant difference (p > 0.05) between RV-PCR and standard culturing methods for detecting the percent survival of spores. RV-PCR results were obtained in < 17 h compared to 7 days for the standard culturing method. High-throughput sample processing and RV-PCR protocols were also developed and tested for synthetic wipe samples containing reference dirt material. RV-PCR protocols allowed processing and accurate analysis of ~ 100 dirty wipe samples (2â³Â à2â³ synthetic) containing ~ 10 viable B. atrophaeus spores in < 24 h. Quantitative RV-PCR protocols based on a Most-Probable-Number (MPN) statistical approach developed for B. anthracis Sterne resulted in more rapid turnaround times than those for traditional culturing and no significant difference in log colony-forming units compared to traditional viability analysis. Integration of RV-PCR assays with high-throughput protocols will allow the processing of 200 wipe samples per day per robot using commercially available automation.
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Authors
S.R. Kane, S.E. Létant, G.A. Murphy, T.M. Alfaro, P.W. Krauter, R. Mahnke, T.C. Legler, E. Raber,