利用多重PCR检测兔肠致病性大肠杆菌和魏氏梭菌的毒力基因

Multiplex PCR for detection of rabbit EPEC and Clostridium Perfringen

我们建立了一种同时检测兔肠致病性大肠杆菌和魏氏梭菌毒力基因的二重PCR方法,在一次PCR反应中同时扩增兔肠致病性大肠杆菌(REPEC)的eae毒力基因和魏氏梭菌的α-toxin毒力基因,扩增产物经电泳,出现与阳性对照细菌条带大小一致的条带即判断为该标本阳性,本试验还对79份腹泻兔粪便或肠内容物样品进行了检测,结果35.4%(28/79)检出eae+的REPEC,而魏氏梭菌的检出率为6.3%。敏感性试验结果表明,该二重PCR能检出10CFU的REPEC,10CFU的魏氏梭菌,具有简便、快速、特异、经济等特点,宜于临床应用。

A multiplex polymerase chain reaction was optimized to simultaneously detect two toxin gene of REPEC and Clostridium Perfringen in this research. Two sets of specific primers were designed according to the sequences of eae and α - toxin gene in the GenBank. It was showed that all samples which contained eae and α - toxin could be amplified by the muhiplex PCR using these two sets primers, yielding two specific, bands of toxin gene of REPEC 833 bp and clostridium α-toxin 324 bp. But no specific band amplified from three other bacteria. 79 weaning rabbit diarrhea samples were also detected by this multiplex PCR. Sensitivity test result confirmed that as little as 10 CFU of REPEC, 10 CFU of Clostridium Perfringen could be detected by this multiplex PCR. This method was also prove to be highly specific , sensitive, universal, rapid, simple as well as economical, and reduce cross contamination for diagnosis of infectious diarrhea.