三对引物同时PCR分型检测产毒素大肠杆菌的方法及在分子流行病学研究中的应用

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

在产毒素大肠杆菌（ＥＴＥＣ）肠毒素基因内设计合成三对引物，建立了三对引物同时ＰＣＲ检测ＥＴＥＣ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，可同时分型检测出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。将建立的方法用于山东省六县市６２３例大肠杆菌致泻标本的检测，阳性率为４０．３％，并能鉴别ＥＴＥＣ的基因型。

A Polymerase Chain Reaction (PCR) method has been developed for detecting Enterotoxigenic Escherichia coli (ETEC). Three different sets of oligonucleotide primer were simultaneously used to amplify the enterotoxin genes of heat-labile (LTh) and heat-stable (STIa and STIb) enterotoxins of ETEC. These primers amplified the 627, 240 or 169 base pair DNA fragments from LTh, STIa and STIb genes of the reference ETEC strains, respectively.Five types of ETEC strains corresponding to the LTh, STIa,STIb,LTh-STIa,or LTh-STIb genotypes were distinguished by a single procedure of PCR, using the mixture of the three sets of primers.There was no cross-reaction with the non-ETEC strains. The lowest detection level was 10 cfu. A total number of 623 stool specimens of diarrheal patients from Shangdong Province induced by E.coli were examined by PCR and the positive rate of ETEC was 40.3%. The results indicated that PCR is a rapid,sensitive and specific method for detecting ETEC.