炭疽杆菌双重实时荧光PCR检测方法的建立

目的 建立同时检测炭疽杆菌capA基因、PA基因的双重实时定量荧光PCR方法,用于应对突发传染病疫情和流行病学调查、突发公共卫生事件应急处置的早期检测及防范生物恐怖威胁.方法 设计和合成分别针对炭疽杆菌capA基因、PA基因的引物对和荧光双标记探针,构建质粒标准品,通过优化探针、引物浓度、反应体系试剂组分等参数,建立可同时检测capA基因、PA基因的双重实时荧光PCR方法,测试方法的灵敏度和特异性,并在实战检测中应用.结果 双重实时荧光定量PCR的炭疽杆菌capA基因、PA基因检测的灵敏度分别可达到每反应5和50拷贝,并具有良好的特异性,在实战应用中亦经过考验.结论 双重实时荧光定量PCR技术具有可以同时筛查、经济、快速、特异性强等优点,在炭疽杆菌检测方面有良好应用价值.

Development of a duplex real-time PCR assay for detection of bacillus anthracis

Objective To develop a dupplex real-time PCR for the detection of specific struetural genes and virulence genes in bacillus anthracis. Methods Two PAirs of specific primers and two fluorogen-labled probes were designed and synthesized according to caPA and PA genes of bacillus anthracis. The reaction PArameters such as the concentration of primers, probes and the reaction buffer were optimized to develop one sets of dupplex real-time PCR assay for the rapid detection of bacillus anthracis. Results The detectable concentration for the duplex real-time PCR were 5 template copy per reaction and 50 template copy per reaction respectively, and had good specificity, stability and reproducibility. Conclusion In this study, the duplex real-time fluorescence quantitative PCR assay indi- cated optimal specificity and sensitivity and deserves to be applied for the detection of bacillus anthracis.