动物源大肠杆菌O157:H7多重PCR检测方法的初步研究

摘要

为建立快速、特异的分离鉴定大肠杆菌O157:H7的多重PCR方法,根据GenBank上已发表的大肠杆菌O157:H7菌体抗原和鞭毛抗原的基因序列(rfbE和fliC基因)设计2对特异引物,分别建立针对rfbE fliC基因的单一PCR方法；通过优化扩增条件,进一步建立可用于动物粪便检测的大肠杆菌多重PCR方法,并进行特异性和灵敏性试验,同时还初步应用到临床样品的检测中.结果表明:所建立的多重PCR方法能同时扩增出rfbE基因(327 bp)和fliC基因( 247 bp)的特异性片段,特异性结果显示其他非大肠杆菌O157:H7的扩增结果均为阴性,表明该方法有较高的特异性；灵敏度试验结果显示细菌纯培养物的检测灵敏度为102cfu/mL.通过试验初步建立了快速、灵敏、特异的检测大肠杆菌O157:H7的多重PCR方法,为从动物粪便中分离鉴定大肠杆菌O157:H7提供了一种简单、灵敏的试验方法,可用于临床动物携带大肠杆菌O157:H7的分子流行病学调查.%To develop a multiplex PCR method for rapid and specific detection of Escherichia coli 0157: H7, two pairs of primers were designed based on the rfbE and fliC genes of Escherichia coli 0157: H7 in GenBank,from which two sets of PCR were established specific to rfbE and fliC gene, respectively. And then they were combined to a multiplex PCR after being optimized. The multiplex PCR system was evaluated for its specificity and sensitivity, and used to detect the clinical isolates. The results showed that the assay could amplify the 327 bp and 247 bp regions of corresponding genes rfbE and fliC. The multiplex PCR method had the characteristics of good specificity and repeatability, the sensitivity being 102 cfu/mL of bacteria samples. The multiplex PCR was a rapid, specific and sensitive method for the detection of Escherichia coli 0157: H7,and provided new method for detection of Escherichia coli 0157: H7.