三对引物同时PCR检测大肠杆菌的方法及在分子流行病学研究中的应用

Study on Molecular Epidemiology of Diarrhea Caused by Enterotoxigenic Escherichia coli by the Plymerase Chain Reaction Using Multiple Primer Pairs

在大肠杆菌（ETEC）肠毒素基因内合成三对引物，建立了三对引物同时PCR检测RTEC的方法，一次PCR即可扩增出627bp（LTh）、240bp（STIa）和169bp（STIb）三种肠毒素基因片段，可同时鉴别LTh、STIa、STIb、LTh－STIa、LTh－STIb五种基因型的ETEC，与非ETEC对照菌无交叉反应，最小检出量为10cfu，显示了很高的特异性和敏感性。摸索出一种简便的腹泻粪便标本处理方法，并应用于山东省六县市ETEC感染的初步调查，为国内ETEC腹泻的分子流行病学研究提供了基本数据和较先进的诊断工具。

A polymerase chain reaction (PCR) method has been developed for the detection of enterotoxigenic Escherichia coli (ETEC). Three different sets of oligonucleotide primers synthesized were used to amplify the enterotoxin genes of heat-labile (LTh) and heat-stable (STI a and STI b) enterotoxins of ETEC. These primers amplified 627, 240 or 169 base pair DNA fragments from LTh, STI a and ST I b genes of the reference ETEC strains,respectively. Five types of ETEC strains corresponding to the LTh, STI a, STI b,LTh-STI a, or LTh-STIb genotypes were distiguished by a single procedure of PCR using the mixture of the three sets of primers. There was no cross-reaction with the nonETEC strains, The lowest detection level was 10cfu. 623 stool specimens of diarrheal patients from Shangdong Province induced by E. coli were examined by the PCR and the positive rates of ETEC were 40.3%. The results indicate the PCR is a rapid, sensitive and specific method for detecting ETEC.