多重PCR方法快速检测4种主要致腹泻性大肠埃希菌

肠毒素性大肠埃希菌(ETEC)、肠致病性大肠埃希菌(EPEC)、肠出血性大肠埃希菌(EHEC)和侵袭性大肠埃希菌(EIEC)是引起腹泻的主要大肠埃希菌,威胁着食品安全和人类健康,建立同时检测4种致腹泻性大肠埃希菌的方法具有重要意义。基于ETEC LT肠毒素基因、EPEC bfpA基因、EHECO抗原基因和EIEC侵袭性质粒特异性基因,设计了4对特异性引物,通过对单一PCR反应条件的优化建立了快速检测4种主要致腹泻性大肠埃希菌的多重PCR方法,彼此之间无交叉反应。该多重PCR方法具有良好的特异性和灵敏性,对24株致病菌进行检测,所试4株致腹泻性大肠埃希菌均为PCR阳性,其他菌株则为阴性。实践证明,利用所建立的多重PCR方法对124份肉类、奶类制品及人工污染样品等进行检测,检出15份阳性,与国标(GB4789.6-1994)检测结果相同。结果表明本文建立的多重PCR方法可用于ETEC、EPEC、EHEC和EIEC的单一或混合感染的鉴别诊断及食品安全风险评估,具有良好的实用性。

Rapid Detection of Four Major Diarrheogenic Escherichia coli by Multi-PCR Method

Enterotoxigenic E.coli （ETEC）, enteropathogenic E.coli （EPEC）, enterohemorrhagic E.coli （EHEC）, and enteroinvasive E.coli （EIEC） are the major E.coli of causing diarrhea and threaten food safety and human health. Therefore, the development of a method to detect the four E.coli simultaneously is of great importance. In this study, four pairs of specific primers were designed according to the gene sequences of ETEC LT, EPEC bfpA, EHEC O-antigen, and EIEC invasion plasmid. A multi-PCR method to detect four kinds of diarrheogenic E.coli was established via optimizing the reaction conditions in single PCR process without cross reaction for each other was detected. The multi-PCR method has good specificity and sensitivity, and 24 bacteria strains were detected using the method, all four kinds of diarrheogenic E.coli were PCR positive result. In practice, 15 positive samples were detected from 124 pieces of meat, dairy products and artificially contaminated samples, which accorded with the testing result by GB （GB 4789.6-1994）. The results indicated that the multi-PCR assay established in this study could be applied in rapid detection for major diarrheogenic ETEC, EPEC, EHEC, and EIEC singly and/or mixed infection diagnoses and food safety risk assessment, it has good practicality.