Rapid Detection Of Yersinia Pestis And Bacillus Anthracis With Real-time PCR Assays

Aim: To develop a simple and specific real-time PCR method for rapid detection and identification of Yersinia pestis and Bacillus anthracis. Methods: Real-time PCR methods with SYBR Green I were developed and optimized using Roche LightCycler Instrument for establishing rapid detection and identification system. The primers were designed based on signature sequence of chromosomal DNA fragment for detection of Y. pestis and on the genes encoding capsule and edema factor in two virulence-related plasmids for detection of B. anthracis. The sensitivity was evaluated and the specificity was confirmed by double-blind method. The reproducibility and the detection limits of spike viscera samples were tested. The primers of internal control were designed based on 16S rDNA sequence of respective bacteria for establishing internal control to avoid false negative. Results: (1) Real-time PCR reaction system was established for detecting Y. pestis. The sensitivity was 1.6 CFU per reaction. The detection results of 28 strains of non-Y. pestis and 275 strains of Y. pestis showed high specificity of this assay. The double-blind examination of qualitative analysis showed the exact specificity. The double-blind examinations of quantitative analysis showed the correlation coefficient was 0.979 and 0.985. For the spike samples, 4000 CFU per reaction system could be detected. (2) Real-time PCR reaction system was established for detecting B. anthracis. The detection results of 29 strains of non- B. anthracis and 14 strains of B. anthracis showed high specificity of this assay. The double-blind examination of qualitative analysis showed the exact specificity. The double-blind examinations of quantitative analysis showed the correlation coefficient was 0.994 and 0.990. The sensitivity for chromosomal DNA detection was 53pg per reaction and for two recombinant plasmids 12 and 140 copies respectively. For the spike samples, 36 spores per reaction system could be detected. Conclusion: The PCR...