巢式PCR快速检测炭疽芽孢杆菌

RAPID DETECTION OF BACILLUS ANTHRACIS BY NESTED PCR

目的 :快速简便地检测炭疽芽孢杆菌。方法 :细菌培养物经反复冻融 ,SDS、蛋白酶 K和煮沸处理后 ,作为PCR模板 ,根据炭疽芽孢杆菌质粒 p X0 1中水肿因子 (EF)基因设计两对引物 ,采用巢式 PCR (nested PCR)扩增目的基因。结果 :从炭疽芽孢杆菌模板中成功扩增出 12 4 7bp的特异片段 ,而未在炭疽芽孢杆菌无毒株、蜡样芽孢杆菌和枯草杆菌模板中扩增出相应条带 ;第一次扩增能检出的最低细菌量是 10 3拷贝 ;经再次巢式 PCR,扩增出 2 0 8bp的特异片段 ,最低检出的细菌数为 10个拷贝 ,敏感性提高了 10 0倍。结论 :巢式 PCR是一种快速、特异。

Objective:To detect Bacillus anthracis by a simple and rapid procedure.Methods:A nested PCR was used to detect Bacillus anthracis spores. Outer and inner pairs of primers were designed from the edema factor(EF) gene of the plasmid pX01. By using lysis methods in preparing the samples for PCR, it was possible to amplify the 1247-bp DNA fragment from samples and the 208-bp DNA fragment from first PCR product.Results:A 1247-bp DNA fragment was amplified from samples of B. anthracis, but not from samples of B. anthracis without toxic plasmid strain, B. cereus and B. calfactor. As few as 10 3 copies of B. anthracis containing EF was detected in first PCR. Subjecting the product of this PCR to a second PCR designed to amplify a 208-bp fragment nested within the 1247-bp product improved detection to 10 B. anthracis copies per PCR.Conclusion:The nested PCR detection method proves to be rapid, sensitive and specific in monitoring of B. anthracis contamination in the environment.