大肠杆菌O8、O9和O89血清型三重PCR检测方法的建立

Development of a triplex PCR method for detection of Escherichia coli O8,O9 and O89 serotypes

为建立一种快速鉴定O8、O9和O89血清型大肠杆菌的三重PCR方法,根据GenBank登录的3种血清型大肠杆菌O抗原合成基因簇序列,设计3对检测引物,通过优化反应条件建立三重PCR方法。结果:经条件优化后的三重PCR方法可有效鉴别O8、O9和O89血清型的大肠杆菌,具有良好的特异性,对其他肉羊养殖场常见血清型的大肠杆菌和致病菌无特异扩增条带;敏感性检测显示,三重PCR方法对O8和O89血清型的最小检出量为10 pg/μL,对O9血清型的最小检出量为100 pg/μL;采用该方法对临床分离的大肠杆菌进行检测,结果与传统血清凝集方法一致。综上,本研究建立的三重PCR方法可快速、准确、特异地对O8、O9和O89血清型大肠杆菌进行检测,为养殖及肉品加工环节大肠杆菌的流行病学研究提供了更加快捷的检测手段。

The aim of this study was to establish a triplex PCR method for rapid identification of Escherichia coli O8,O9 and O89 serotypes.Three pairs of primers targeting the O antigen synthesis gene cluster of E.coli O8,O9 and O89 serotypes were designed,respectively.A triplex PCR method was developed after optimizing the reaction conditions,and then the sensitivity and specificity of the triplex PCR was examined.Clinical E.coli isolates were detected by triplex PCR to test the practicability of the method.The results showed that the optimized triplex PCR method could effectively identify O8,O9 and O89 serotypes of E.coli.However,there was no specific amplification band for the common serotypes of E.coli and other pathogenic bacteria in the specificity test.The sensitivity test results showed that the minimum detection amount of the triplex PCR method for O8,O9 and O89 serotypes were 10 pg/μL,100 pg/μL and 10 pg/μL,respectively.The clinically isolated E.coli strains were detected by this method,and the results were consistent with those of the traditional serum agglutination method.In conclusion,the triplex PCR established in this study could detect the O8,O9 and O89 E.coli serotypes more quickly,accurately and specifically,which would serve as a useful and rapid detection method for the epidemiological study of E.coli in the animal breeding and meat processing industries.