Rapid, high-throughput, culture-based PCR methods to analyze samples for viable spores of Bacillus anthracis and its surrogates. Export

@article{citeulike:4007842, 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 (10(6)). Field tests using approximately 1000 biological indicators, each containing 10(6) 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 (2''x2'' 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.}, author = {Kane, S R R. and L\'{e}tant, S E E. and Murphy, G A A. and Alfaro, T M M. and Krauter, P W W. and Mahnke, R and Legler, T C C. and Raber, E}, citeulike-article-id = {4007842}, citeulike-linkout-0 = {http://dx.doi.org/10.1016/j.mimet.2008.12.005}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/19141303}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=19141303}, day = {24}, doi = {10.1016/j.mimet.2008.12.005}, issn = {0167-7012}, journal = {Journal of microbiological methods}, keywords = {counting, mpn}, month = {December}, posted-at = {2009-02-04 15:16:25}, priority = {2}, title = {Rapid, high-throughput, culture-based PCR methods to analyze samples for viable spores of Bacillus anthracis and its surrogates.}, url = {http://dx.doi.org/10.1016/j.mimet.2008.12.005}, year = {2008} }

TY - JOUR ID - citeulike:4007842 L3 - citeulike-article-id:4007842 N2 - 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 (10(6)). Field tests using approximately 1000 biological indicators, each containing 10(6) 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 (2''x2'' 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. JF - Journal of microbiological methods SN - 0167-7012 TI - Rapid, high-throughput, culture-based PCR methods to analyze samples for viable spores of Bacillus anthracis and its surrogates. KW - counting KW - mpn AU - Kane, S R AU - Létant, S E AU - Murphy, G A AU - Alfaro, T M AU - Krauter, P W AU - Mahnke, R AU - Legler, T C AU - Raber, E PY - 2008/12/24/ UR - http://dx.doi.org/10.1016/j.mimet.2008.12.005 ER -