Detection Of Francisella Tularensis And Bacillus Anthracis With Real-time PCR Assays

Aim: Francisella tularensis and Bacillus anthracis, which are highly pathogenic, have the most potential to be misused as the pathogens for biological weapon and bioterrorism agents, posing a serious threat to human health. Therefore, it is urgent to develop rapid approach for specific and sensitive detection of Francisella tularensis and Bacillus anthracis.Methods: Based on the Roche LightCycler and TaqMan technology, we developed and optimized the real time PCR methods for rapidly detecting and identifying these two pathogens. For detecting Francisella tularensis, we designed TaqMan primers and fluorescent probes against specific sequences within the chromosome of this pathogen. Bacillus anthracis was identified by multiple TaqMan primers and fluorescent probes against specific sequences (GS sequences) within the chromosome as well as genes encoding the capsule and lethal factor located on the virulence-related plasmids. The specificity and sensitivity of these assays were evaluated, respectively. Strains with high homology at DNA level were selected for evaluating the specificity of the assays; recombinant plasmid or attenuated strains and genomic DNA were used as templates to evaluate the sensitivity of the assays. The feasibility of these assays in quantitative detection of target genes in soil samples was evaluated using spiked soil samples with different concentrations of recombinant E.coli or attenuated strain. Theses assays were also compared with the imported real time PCR kits in the mobile vehicle with functions of nuclear, chemical and biological detection.Results: (1) The TaqMan real-time PCR assays were developed based on AKR and fopA genes for detecting Francisella tularensis. Non-specific amplifications were not found from non-Francisella tularensis templates. The sensitivity for detection of recombinant plasmid was 10 copies per reaction; when Francisella tularensis genomic DNA was used as a template, with AKR-F/R primers, the sensitivity could achieve 0.25pg/μl; with fopA-F/R primers, 2.5pg/μl, and based on AKR-F/R and fopA primers, 440 CFU and 960 CFU bacteria per gram spiked soil could be detected, respectively. A blind detection of the target genes in the spiked soil samples demonstrated the correct quantitative detection within the range of sensitivity. (2) The TaqMan real-time PCR assays were developed based on GS sequences within Bacillus anthracis chromosome and the genes encoding capsule and lethal factor on two virulence-related plasmids for detecting Bacillus anthracis, whereas non-specific amplifications were not detected from non-Bacillus anthracis templates. Using plasmid DNAs of BA-lef, BA-capA and BA-GS recombinant strains as a template, with capA-F/R and GS-F/R primers, the sensitivity for each reaction was 10 copies, with lef-F/R primer, the result was 102 copies for each reaction.Using Bacillus anthracis Sterne strain as template, with Lef-F/R and GS-F/R primers, the detection sensitivity was 1.2×10-2 CFU/μl. Using Bacillus anthracis genomic DNA as template, with lef-F/R primers, the sensitivity could achieve 2.4fg/μl, with capA-F/R primers, 24 fg/μl, and with GS-F/R primers, 0.24pg/μl.Using anthrax spores of Sterne strain spiked in soil samples, with Lef-F/R and GS-F/R primers, the sensitivity was up to 2×103 CFU bacteria per gram spiked soil. A blind detection of the target genes in the spiked soil samples demonstrated the correct quantitative detection within the range of sensitivity. The parallel comparison with the imported real time PCR kits for detecting Bacillus anthracis and Francisella tularensis proved that our real time PCR reagents were comparable to the imported ones interms of sensitivity and specificity.Conclusion: The TaqMan real-time PCR assays established in this study were sensitive and specific for rapid identification and detection of Francisella tularensis and Bacillus anthracis. These assays could be completed within 1h, providing a powerful tool for rapid detection and identification of pathogens during clinical diagnosis, environmental monitoring, biological control, and public health emergencies.