多重PCR-DHPLC检测4种主要致腹泻性大肠埃希氏菌方法的建立与应用  

Development and Application of a Multiplex PCR/DHPLC Method for Detecting Four Common Diarrheogenic Escherichia coli Strains

作　　者：徐义刚 崔丽春[2] 李苏龙 曹际娟[3] 姜艳春 张子群 刘新亮 

机构地区：[1]黑龙江出入境检验检疫局检验检疫技术中心,黑龙江哈尔滨150001 [2]东北林业大学,黑龙江哈尔滨150040 [3]辽宁出入境检验检疫局,辽宁大连116065

出　　处：《食品科学》2010年第20期293-297,共5页Food Science

基　　金：黑龙江省青年基金项目(QC2009C90);黑龙江省博士后基金项目(LBH-Z09)

摘　　要：肠出血性大肠杆菌(EHEC)、肠致病性大肠杆菌(EPEC)、产肠毒素性大肠杆菌(ETEC)和肠侵袭性大肠杆菌(EIEC)是引起腹泻、危害食品安全和人类健康的4种主要大肠杆菌。本实验基于ETEC LT基因、EPEC bfpA基因、EHEC O抗原基因和EIEC侵袭性质粒基因序列,设计了4对特异性引物,利用聚合酶链反应(PCR)和变性高效液相色谱(denaturing high performance liquid chromatography,DHPLC)技术,通过体系优化,建立了致腹泻性大肠杆菌多重PCR-DHPLC检测方法,达到同时检测4种致病菌的目的。该方法灵敏度高,最低检测限为EHEC6×101拷贝/μL、ETEC1.3×102拷贝/μL、EPEC9×101拷贝/μL、EIEC7×101拷贝/μL。特异性试验表明,对22种共41株细菌进行检测,所试6株致腹泻性大肠杆菌均为阳性。实践证明,该方法具有良好的实用性,可用于ETEC、EPEC、EHEC和EIEC的单一或混合感染的检测。Four pairs of specific primers were designed according to the sequences of the LT gene from enterotoxigenic E.coli （ETEC）,the bfpA gene from enteropathogenic E.coli （EPEC）,the O antigen gene from enterohemorrhagic E.coli （EHEC） and the invasive plasmid gene from enteroinvasive E.coli （EIEC） for the development of a multiplex polymerase chain reaction/denaturing high performance liquid chromatography （PCR/DHPLC） method for the simultaneous detection of the four common diarrheogenic E.coli strains. The PCR/DHPLC method had high sensitivity and its detection limit was 6 × 101 copies/μL for EHEC,1.3×102 copies/μL for ETEC,9×101 copies/μL for EPEC and 7×101 copies/μL for EIEC. The specificity evaluation showed that 6 of 41 stains from 22 species were determined to be positive. This method proves to be applicable and suitable for the detection of infection with one or more of the four E.coli strains.

关 键 词：致腹泻性大肠杆菌 聚合酶链式反应 变性高效液相色谱 检测 

分 类 号：R155.1[医药卫生—营养与食品卫生学]