title: Rapid, high-throughput, culture-based PCR methods to analyze samples for viable spores of Bacillus anthracis and its surrogates.
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 approximately100 dirty wipe samples (2''x2'' synthetic) containing approximately10 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.
authors.affiliation: Lawrence Livermore National Laboratory, Livermore, CA 94550, United States. kane11@llnl.gov <kane11@llnl.gov>
authors.forename: S R
authors.initials: SR
authors.lastname: Kane
authors.forename: S E
authors.initials: SE
authors.lastname: Létant
authors.forename: G A
authors.initials: GA
authors.lastname: Murphy
authors.forename: T M
authors.initials: TM
authors.lastname: Alfaro
authors.forename: P W
authors.initials: PW
authors.lastname: Krauter
authors.forename: R
authors.initials: R
authors.lastname: Mahnke
authors.forename: T C
authors.initials: TC
authors.lastname: Legler
authors.forename: E
authors.initials: E
authors.lastname: Raber
mesh_terms: D001331:Automation; D001408:Bacillus anthracis / Q000302:isolation & purification*; D001431:Bacteriological Techniques; D017606:Chlorine Compounds; D004784:Environmental Monitoring / Q000379:methods; D050296:Microbial Viability; D010087:Oxides; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013171:Spores, Bacterial / Q000302:isolation & purification
chemical_list: D017606:Chlorine Compounds; D010087:Oxides; C025109:chlorine dioxide