肠出血性大肠埃希菌O157∶H7环介导恒温扩增快速检测方法的建立与应用

Establishment of rapid method for detecting enterohemorrhagic E.coli O157∶H7 by loop-mediated isothermal amplification

目的肠出血性大肠埃希菌(Enterohemorrhagic Escherichia coli,EHEC)O157∶H7是一种重要的人畜共患病致病菌,主要通过被污染的食物传播,引起出血性结肠炎,建立其快速检测方法具有重要意义。方法基于EHEC O157∶H7保守的rfbE基因序列,设计4条引物,利用环介导等温扩增技术(loop-mediatedisothermal amplification,LAMP),成功建立了EHEC O157∶H7LAMP快速检测方法 ,60min内即可完成致病菌检测。结果利用该LAMP方法对30种共38株细菌进行检测,所试EHEC O157∶H7均为阳性结果 ,说明该方法具有高度特异性。本方法对纯培养的EHEC O157∶H7检测限为12CFU/mL,污染食品中EHEC O157∶H7的检测限为18CFU/g。实践应用表明,对1121份进出口肉类、奶类制品及人工污染样品等进行检测,检出57份LAMP阳性,与采用AOAC标准检测结果的符合率为100%。结论该LAMP方法操作简便、特异性强、灵敏度高,具有良好的实用性。

Enterohemorrhagic E.coli（EHEC）O157∶H7 is an important pathogen of zoonose,mainly initiates its transmission through contaminated food and causes hemorrhagic colitis.Thus,development of rapid method for detecting EHEC O157∶H7 is of great importance.According to highly conserved rfbE gene of EHEC O157∶H7,four primers were designed,then the method for detecting EHEC O157∶H7 was established by DNA loop-mediated isothermal amplification（LAMP）,which could finish the test in 60 min.Thirty-eight bacteria strains were tested by the LAMP method and only EHEC O157∶H7 strains were LAMP positive results,indicating the high specificity of the method with detection limit of 12 CFU/mL for cultivated EHEC O157∶H7 and of 18 CFU/g for its contaminated food.In practice,57 portions of positive samples were detected from 1 121 portions of meat,dairy products and artificial contamination samples by the LAMP method,which accorded with the detection result from AOAC standard.It＇s indicated that the LAMP assay established in this study is a sensitive,rapid and simple tool for the detection of EHEC O157∶H7.