动物源性肠出血性大肠杆菌O157∶H7及其3个毒力基因的多重PCR快速检测研究  

Detection of animal-derived Escherichia coli O157∶H7 and its three virulence genes by multiplex PCR technique

作　　者：陈雅君[1,2] 王亚宾[1] 张莉娟[1] 张龙现[1,2] 陈丽颖[1] 刘中原[1] 申果[1] 胡慧[1,2] 

机构地区：[1]河南农业大学牧医工程学院,郑州450002 [2]河南省动物性食品安全重点实验室,郑州450002

出　　处：《中国人兽共患病学报》2013年第7期686-691,共6页Chinese Journal of Zoonoses

基　　金：河南省重大公益科研项目(81100912300);漯河市科技计划项目(081203)~~

摘　　要：目的建立快速、特异的分离鉴定大肠杆菌O157∶H7及其3个主要毒力基因(hylA、eaeA和stx2基因)的多重PCR方法。方法根据GenBank公布的大肠杆菌O157∶H7菌体抗原rfbE基因、鞭毛抗原fliC基因、溶血素(hlyA)基因、紧密黏附素(eaeA)基因和志贺样毒素2(stx2)基因为靶基因,设计5对特异性引物,在同一扩增体系中进行PCR,优化反应体系,测定特异性和灵敏度,并进行了临床样品的检测。结果该方法扩增目的基因片段分别为327bp、247bp、494bp、384bp和779bp,特异性和灵敏度均高,细菌纯培养物的检测灵敏度为104cfu/mL。结论初步建立了快速、灵敏、特异的检测肠出血性大肠杆菌O157∶H7及其3个毒力基因的多重PCR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及食品微生物检测。A multiplex PCR method was developed for rapid and specific detection of Escherichia coli O157︰H7 and three of its virulence genes （hylA, eaeA and stx2 gene）.Five sets of primers were designed according to the sequences of rfbE gene、fliC gene、hemolysin gene（hlyA）、inthnin gene （eaeA） and shiga-like toxin gene 2（stx2）of E.coli O157 were selected and added into one amplification system to perform PCR,The system was optimized, the specificity and sensitivity of this system were evaluated, and used to detect the clinical isolates. Results： The assay was designed to amplify the 327 bp、247 bp、494 bp、384 bp、and 779 bp regions of corresponding genes rfbE、fliC、hlyA、eaeA and stx2. It was sensitive and specific highly. Sensitivity of the assay was 104cfu /ml of bacteria samples. Conclusions： A rapid, specific, and sensitive multiplex PCR technique for the simultaneous detection of E.coli O157︰H7 and three of its virulence genes has been studied primarily. And available to test for the quantitative detection of E.coli O157︰H7 from the contaminated food samples.

关 键 词：动物源性 肠出血性大肠杆菌O157∶H7 毒力基因 多重PCR 

分 类 号：S855.1[农业科学—临床兽医学]