多重PCR方法检测大肠杆菌O157∶H7的初步研究

Detection of Escherichia coli O157:H7 by multiplex polymerase chain reaction

目的研究一种快速、特异的检测大肠杆菌O157∶H7的多重PCR方法。方法以大肠杆菌O157∶H7的O157抗原基因(rfbE基因)、鞭毛H7抗原基因(fliC基因)、粘附素基因(eaeA基因)和志贺样毒素Ⅰ、Ⅱ(SLT-Ⅰ、SLT-Ⅱ基因)为扩增对象,设计能导至目的片段大小不同的5对引物,通过优化条件,建立了可用于粪便检测的大肠杆菌5重PCR。结果在同一扩增体系中,检测了5株不同来源的大肠杆菌O157∶H7及27株非大肠杆菌O157∶H7细菌。结果表明,该方法能从2株大肠杆菌O157∶H7中扩增出rfbEf、liC、eaeA、SLT-Ⅰ、SLT-Ⅱ基因,它们的大小分别为582 bp、802 bp、487 bp3、68 bp和177 bp。而另外3株大肠杆菌O157∶H7也能扩增出除eaeA、SLT-Ⅱ基因外的其它3个基因片段。表明该方法可用于检测大肠杆菌O157∶H7,还可检测细菌是否含有毒素基因。在检测27株非大肠杆菌O157∶H7,除O149出现SLT-II扩增产物和O1∶H7出现fliC基因扩增产物外,其它非大肠杆菌O157∶H7的扩增结果均为阴性,表明该方法有较高的特异性。当粪便样品经增菌培养12 h,用该5重PCR体系能检测出最初接种量为5cfu/g粪便的样品。结论本5重PCR方法具有较高的特异性和敏感性,可迅速、有效地将大肠杆菌O157∶H7与其它非大肠杆菌O157∶H7相区别,同时还能检测O157∶H7中的毒力因子。因此,通过进一步研究,本方法有望应用于临床大肠杆菌O157∶H7感染的辅助诊断。

To develop a quick and sensitive multiplex polymerase chain reaction （PCR） for the detection of Escherichia coli O157 ： H7, 5 pairs of primers were designed,in which the segments of the O157 gene（rfbE）, flagella antigen H7 gene （fliC）, adhesion gene（eaeA）, Shiga toxin Ⅰ and Ⅱgenes（slt-Ⅰ and Slt-Ⅱ） were specifically amplified and a novel method of multiplex PCR assay was developed. This method allowed to identify simultaneously the serotype O157 ： H7 and its virulent factors by using a single reaction. Among 32 strains of bacteria analyzed, 5 belonged to E. coli O157 ： H7, and 27 to non-O157 ： H7. In addition, this multiplex PCR assay could also distinguish serotype O157 ： H7 from other serotypes of E. coli and other bacteria, and did not cross-react with the background bacteria in cattle feces. Also, this method could detect the presence of E. coli O157 ： H7 as few as the initial inoeulum of 5 cfu/g of 12-hours enrichmentcuhures in samples. It is concluded that the multiplex PCR assay is highly specific and sensitive, and can be used to detect the presence of E. coli O157 ： H7 and its virulent factor.