猪源产肠毒素大肠杆菌三重PCR检测方法的建立及应用

Development and Application of triplex PCR Detection of porcine Enterotoxigenic E.coli

为建立一种简单、快速、灵敏、准确的产肠毒素大肠杆菌(ETEC)检测方法,根据产肠毒素大肠杆菌菌毛(K88)和毒素(STa和LT)基因分别设计合成了1对引物,对K88、STa和LT基因扩增条件进行优化,建立了检测K88、STa和LT的三重PCR方法。该方法对K88、STa和LT基因的扩增产物分别为499 bp,190 bp和373 bp;此外,该方法具有良好的灵敏性和特异性。本实验建立的三重PCR方法为致幼畜腹泻ETEC的检测提供了快速准确方法。用所建立的三重PCR方法对实验室从临床腹泻样品中分离的120株大肠杆菌进行检测,结果 9株为K88/LT/STa阳性,14株为K88/LT阳性,21株K88/STa阳性,13株LT/STa阳性,8株K88阳性,2株LT阳性,12株STa阳性。

To establish a simple,sensitive,accurate and rapid method for the detection of Enterotoxigenic E.coli（ETEC）inpigs,a triplex PCR method was developed based on 3 pairs of primers for the fimbriae genes（K88）and 2 toxin genes（STa,LT）am-plification. The reference E.coli strains which passed the different virulence genes were detected. The results showed that 499 bp,190 bp and 373 bp DNA fragments of K88,STa and LT genes were amplified by this protocol,respectively. The triplex PCR wasproved to be specific and sensitive. Furthermore,a total of 120 E.coli were tested and theresults showed that 9 samples were K88/LT/STa positive,14 sample was K88/LT positive,21 sample was K88/LT positive,13 sample was K88/STa positive,8 sample wasK88 positive,2 sample was LT and 12 sample was STa positive. The triplex PCR assay provides a method to detect ETEC whichcauses diarrhea in piglets.