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.If a biothreat agent was released, hundreds to thousands of environmental samples of diverse types would need to be rapidly processed and analyzed in order to first characterize the contamination of the site and then assess the effectiveness of decontamination activities. Decision-makers also need rapid results for remobilizing disinfection equipment in the case of incomplete decontamination and for reopening facilities and areas based on results from clearance sampling (12)(13)(14).Current methods used by the Centers for Disease Control and Prevention (CDC) to assess the viability of spores on surfaces rely on culturing samples on solid media (5, 6). These methods involve several manual steps, including pipetting to prepare dilution series, plating of numerous replicates for a series of dilutions, and colony counting, which make it labor-, space-, and time-intensive. Typically, only 30 to 40 samples may be processed each day with confirmed results obtained days later (5, 6). Validated rapid-viability test protocols are therefore needed to ensure public safety and to help mitigate impacts due to facility closures following a biothreat agent release. This critical need was highlighted during the response to the 2001 anthrax attacks, in which clearance sampling and analysis required excessive time prior to facilities reopening.Because risk assessment after such an attack is determined on the basis of the presence of viabl...

In silico and in vitro evaluation of PCR-based assays for the detection ofBacillus anthracischromosomal signature sequences

“…44 Results of the ring trial confirmed the results obtained in the in silico analysis ( Table 4). The three assays with highest in silico specificity (BA5345, 21 PL3, 47 and BA5357 46 ) all performed well in the ring trial, with diagnostic sensitivity and specificity values close to 1 ( Table 5). Furthermore, these assays were found to be robust and provided consistent results between laboratories (kappa values of 0.9-1.0).…”

“…30 These genetic markers provide limited specificity and require additional timeconsuming and labor-intensive post-PCR analysis steps. Other areas of the chromosome have also been investigated as potential DNA-targets for identification purposes, including the so-called BA813 [31][32][33][34][35][36][37][38] and BA5510 sequences, 19 genes bclB, 39 sap, 40,41 saspB, 5,42 and sspE, 22,43 the B-type small acid-soluble spore protein gene (SASP), 44 a glycosyltransferase group 1 family protein, 45 a protein showing similarities with an abhydrolase, 18 and several DNA loci located on prophage regions, 17 i.e., BA5345, 21 BA5357, 46 and PL3. 47 Although most of these regions have been claimed to be anthrax-specific, B. cereus strains sometimes yield false positive results.…”

“…These assays target the markers BA5345, 21,65 PL3, 47 and BA5357, 46 respectively. Three of these assays are based on hydrolysis probe ("TaqMan assay"); the fourth uses SYBR Green chemistry.…”

“…Six published PCR-assays targeting different B. anthracis chromosomal markers were evaluated in vitro. The most specific methods according to in silico analysis 21,46,47 were compared with the assays recommended by the WHO 40,44 and a single assay targeting BA813. 35 Ninety blinded DNA samples were exchanged between partners and an IAC was distributed.…”

Swab Protocol for Rapid Laboratory Diagnosis of Cutaneous Anthrax

“…Since the 2001 bioterrorism-associated anthrax outbreak, several different types of real-time PCR assays for the detection of B. anthracis have been developed (11,12,24,28). The molecular beacon-based assay utilized in this study was selected to assess whether recovery of B. anthracis with the three swab extraction methods would be consistent irrespective of the type of real-time PCR assay used.…”