产气荚膜梭菌毒素型菌落多重PCR方法的建立及应用

Establishment and Application of Colony Multiplex PCR Assay for Toxinotyping Clostridium perfringens

根据产气荚膜梭菌α、β、ε和ι毒素基因cpa、cpb、etx、iA设计并合成4对特异性引物,建立快速鉴定产气荚膜梭菌毒素型的多重PCR方法。结果显示:产气荚膜梭菌A、B、C、D和E型参考菌株均扩增出相应的片段,而肉毒梭菌、气肿疽梭菌、腐败梭菌、诺维梭菌的扩增均为阴性;将产气荚膜菌株单个菌落稀释100倍,仍能扩增出相应的目的片段。利用此多重PCR方法对16株不同动物来源的产气荚膜梭菌进行分型鉴定,并与毒素中和试验鉴定结果进行比较,结果显示2种方法具有较高的符合率。该方法可有效进行产气荚膜梭菌的快速检测和分型,对产气荚膜梭菌感染与食品安全问题的研究具有参考价值。

According to the genes（cpa,cpb,etx,and iA） encoding four major toxins（α、β、ε,τ） of C. perfringens,four primers were designed and the colony multiplex PCR were established. The expected sequences were obtained successfully from C. perfringens reference strains including A, B, C, D and E by the multiplex PCR assay. However, the sequences were not amplified from C. botulinum, C. chauvoei,C, septicum and C. novyi. The expected sequences were obtained from C. perfringens single colony when diluted to 100 times. Sixteen C. perfringens strains isolated from different animals were toxinotyped by the multiplex PCR assay,and the results were compared with the results of toxins neutranization test in mice. The two assays showed good accordance. The established colony multiplex PCR can rapidly detect and toxinnotype C. perfringens effectively,which can be referred for the future study on C. perfringens infection in animal and food safety issue.