产肠毒素大肠杆菌多重PCR检测方法的建立

Multiplex PCR for detection of virulence gene of enterotoxigenic Escherichia coli

为快速检测和鉴定产肠毒素大肠杆菌(ETEC)菌毛(K88和K99)和毒素(STa)基因,本研究设计合成了针对K88、K99和STa基因的3对特异性引物,对K88、K99和STa基因扩增条件进行优化,建立了检测K88、K99和STa的多重PCR方法。该方法对K88、K99和STa基因的扩增产物分别为237 bp,314 bp和166 bp;此外,该方法具有良好的灵敏性和特异性。本实验建立的多重PCR方法为致幼畜腹泻ETEC的检测提供了快速准确方法。用所建立的多重PCR方法对实验室分离的23株大肠杆菌进行检测,结果 2株为K99/STa阳性,1株为STa阳性。

To detect and identify the 2 fimbriae genes （K88, K99） and toxin genes of heat-stable enterotoxin a （STa） in enterotoxigenic Escherichia coli （ETEC）, a multiplex PCR method was developed based on 3 pairs of primers for K88, K99 and STa gene amplification. The reference E.coli strains which passed the different virulence genes were detected and the results demonstrated that 237 bp, 314 bp and 166 bp DNA fragments of K88, K99 and STa genes were amplified by this protocol, respectively. The multiplex PCR was proved to be specificity and sensitivity. Furthermore, a total of 23 E.coli were tested and the results showed 2 samples were K99/STa and 1 sample was STa positive. The multiplex PCR assay provides a method to detect ETEC which caused diarrhea in piglets.