title: Rapid-viability PCR method for detection of live, virulent Bacillus anthracis in environmental samples.
abstract: In the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor- and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulent Bacillus anthracis, in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for the B. anthracis chromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulent B. anthracis in environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples.
authors.affiliation: Lawrence Livermore National Laboratory, Livermore, CA 94550, USA. letant1@llnl.gov
authors.forename: Sonia E
authors.initials: SE
authors.lastname: Létant
authors.forename: Gloria A
authors.initials: GA
authors.lastname: Murphy
authors.forename: Teneile M
authors.initials: TM
authors.lastname: Alfaro
authors.forename: Julie R
authors.initials: JR
authors.lastname: Avila
authors.forename: Staci R
authors.initials: SR
authors.lastname: Kane
authors.forename: Ellen
authors.initials: E
authors.lastname: Raber
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Bunt
authors.forename: Sanjiv R
authors.initials: SR
authors.lastname: Shah
mesh_terms: D001331:Automation / Q000379:methods; D001408:Bacillus anthracis / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D001431:Bacteriological Techniques / Q000379:methods*; D041963:Bacteroidetes; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004783:Environmental Microbiology; D057166:High-Throughput Screening Assays / Q000379:methods; D050296:Microbial Viability; D010957:Plasmids; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors
chemical_list: D004269:DNA, Bacterial