O_(157)∶H_7型大肠杆菌荧光定量PCR检测方法的建立

Establishment of fluorescent quantitative PCR assay for detection of Escherichia coli O_157∶H_7

根据GenBank中登录的肠出血性大肠杆菌保守基因序列Stx1、Stx2设计合成荧光定量PCR引物和探针,对荧光定量PCR的反应条件进行优化,建立了TaqMan荧光定量PCR检测方法。用建立的检测方法对临床分离病料进行了检测,并与常规PCR方法的检测结果做了对比。结果显示,建立的TaqMan荧光定量PCR方法的灵敏度达7.06×103CFU/mL,比常规PCR方法的灵敏度高106倍。用该方法和常规PCR对8种样品进行了检测,证实,该方法与常规PCR的阳性符合率为100%,具有较好的重复性。

Probes and primers for a TaqMan quantitative PCR were designed and synthesized according to the conserved gene sequence Stxl and Stx2 of enterohemorrhagic E. coli（EHEC）, available in GenBank. Then reaction parameters were optimized to develop a TaqMan quantitative PCR assay. The clinical tissue samples were examined by using the established TaqMan quantitative PCR and the results were compared with that by conventional PCR. The established PCR could detect 7.06 ×10^3CFU/mL plasmid DNA and its sensitivity was 1×10^6 times higher than that of the conventional PCR. Eight samples were .used to examine the gene Stxl by the established PCR and conventional PCR and the results indicated that the positive rate for Stxl gene by the established PCR was in accordance with that by the conventional PCR.