产气荚膜梭菌多重PCR定型方法的建立及其应用

Establishment of a multiplex PCR method for Clostridium perfringens typing and its application

建立了多重PCR检测产气荚膜梭菌α、β、ε和ι毒素基因的方法。该方法具有良好的特异性和敏感性,只有产气荚膜梭菌呈现阳性,被检验的其他梭菌以及大肠杆菌、葡萄球菌均为阴性;将肉汤菌液样品10倍系列稀释后进行检测,检测灵敏度达到1.2×104CFU/mL。收集40份牛粪便样品,进行PCR检测,32份样品中成功扩增出589 bp的α毒素基因片段,阳性率为80%。结果显示,建立的多重PCR检测方法可取代血清中和试验,用于产气荚膜梭菌分型,同时表明A型产气荚膜梭菌在当地奶牛场中较为普遍。

A multiplex PCR assay was developed to detect the alpha, beta, epsilon, and iota toxin genes of Clostridium perfringens. The specificity and sensitivity of the PCR method were higher. The specific fragments could be amplified from C. perfringens, but no products were obtained from other Clostridium strains, Escherichia coli and Staphylococcus. The sensitivity of the PCR assay was 1.2×10^4 CFU/mL. Forty samples of cattle feces were collected for PCR detection, and 32 were positive. The positive rate was 80%. The results indicated that neutralization test could be replaced by the new method, and that C. perfringens type A was the most prevalent type in cattle in Qinghai.