产气荚膜梭菌cpe^+菌株的筛选与鉴定

Screening and identification of the cpe^+ strain of Closteidium perfringens

参考相关资料,针对产气荚膜梭菌肠毒素基因(cpe)设计合成1对特异性引物,对34株贵州分离株进行PCR扩增,并将扩增产物连接到pMD18-T载体,转化至大肠杆菌DH5α感受态细胞,提取质粒进行PCR和双酶切鉴定后测序和分析。结果在34株产气荚膜梭菌贵州分离株中有1株C型菌株扩增出与预期大小相一致的目的片段,经克隆测序后,该片段大小为233 bp,其与产气荚膜梭菌参考株肠毒素基因序列的核苷酸同源性为99.6%~100%,推导氨基酸同源性为98.7%~100%,表明所扩增产物为产气荚膜梭菌的肠毒素基因片段。本试验筛选鉴定出1株产气荚膜梭菌cpe+菌株,为今后进一步探讨产气荚膜梭菌性食物中毒机制奠定了基础。

A pair of primers were designed and synthesized according to the Clostridium perfringens enterotoxin gene （cpe） sequences in GenBank. Thirty-four C. perfringerts isolates from Guizhou province were detected by PCR. The PCR products were cloned into pMD18-T vector and transformed into competent cell DH5α. The recombinant plasmid was identified by PCR and restriction enzyme digestion, and then sequenced. The results showed that one of the 34 isolates had the target fragment. The gene sequence consisted of 233 bp, which shared 99.6% - 100% of sequence identity and 98. 7% - 100% of amino acid sequence identity with those of the reference strains in GenBank. The cpe^＋ strain of C. perfringens is a material base for investigating the mechanism of food poisoning caused by this bacterium.