基于TaqMan探针荧光PCR检测致泻性大肠埃希氏菌  

Detection for Diarrheagenic Escherichia coli (DEC) by Real-time PCR with TaqMan Probe

作　　者：袁慕云 许龙岩 柯碧霞[2] 陈瑶[3] Yuan Muyun;Xu Longyan;Ke Bixia;Chen Yao(Guangzhou Customs Technology Center,Guangzhou 510623;Guangdong Provincial Center for Disease Control and Prevention,Guangzhou 510440;Southern Medical University,Guangzhou 510515)

机构地区：[1]广州海关技术中心,广州510623 [2]广东省疾病预防控制中心,广州510440 [3]南方医科大学,广州510515

出　　处：《中国食品学报》2021年第3期274-281,共8页Journal of Chinese Institute Of Food Science and Technology

基　　金：广东省协同创新与平台环境建设专项(2017A040405043)。

摘　　要：建立快速检测致泻性大肠埃希氏菌的(DEC)方法,根据DEC的uidA(GenBank:JQ068101.1)、bfpB(Sequence:NG_034726.1)、eaeA(GenBank:AJ579305.1)、LT(GenBank:M17873.1)、ST(GenBank:M18346.1)、Stx1(GenBank:FR875155.1)、Stx2(GenBank:AB030484.1)、ipaH(GenBank:JQ638638.1)、aggR(GenBank:Z32523.1)设计引物和探针,建立基于TaqMan探针荧光PCR检测方法。用uidA基因检测所有DEC,A体系4个基因bfpB、eaeA、LT、ST检测EPEC和ETEC,B体系4个基因ipaH、Stx1、Stx2、aggR检测EIEC、STEC和EAEC,两个体系探针的5′端分别标记FAM、VIC、TET和ROX。试验结果表明,两个体系扩增效率89.6%~102.2%,线性相关系数R2在0.965~0.999范围,可准确鉴别出25株DEC型别,而70株非DEC扩增结果阴性。本研究所建立方法的特异性强,可准确检测EPEC、ETEC、EIEC、STEC和EAEC。Object:To establish a method for rapid detection of diarrhea-causing Escherichia coli(DEC).Methods:Primers and probes were designed based on uidA(GenBank:JQ068101.1),bfpB(Sequence:NG_034726.1),eaeA(GenBank:AJ579305.1),LT(GenBank:M17873.1),ST(GenBank:M18346.1),Stx1(GenBank:FR875155.1),Stx2(GenBank:AB030484.1),ipaH(GenBank:JQ638638.1),aggR(GenBank:Z32523.1)of DEC to establish a TaqMan probe fluorescent PCR assay.Among these targets,uidA gene was used to identify all DEC,and the remaining targets are divided into two systems,A and B.A system including 4 genes(bfpB,eaeA,LT,ST)were used to detect EPEC and ETEC,B system includes 4 genes(ipaH,Stx1,Stx2,and aggR)were used to detect EIEC,STEC and EAEC.The 5'ends of the two system probes were labeled with FAM,VIC,TET and ROX respectively.Results:The amplification efficiency of the two systems was 89.6%-102.2%respectively and the linear correlation coefficient R2 was in the range of 0.965-0.999.This assay is specific to Escherichia coli(25/25),and the Real-time PCR detection showed that EPEC,ETEC,EIEC,STEC and EAEC could be identified and distinguished by system A and B while 70 non-DEC amplification results were negative.Conclusion:This new method is highly specific to E coli and can accurately distinguish EPEC,ETEC,EIEC,STEC and EAEC.

关 键 词：致泻大肠埃希氏菌 荧光PCR 致病因子 检测 

分 类 号：R446.5[医药卫生—诊断学]