羊源产气荚膜梭菌不同毒素型多重PCR检测方法的建立及初步应用  

Establishment and preliminary application of multiplex PCR for detecting toxintyping of goat Clostridium perfringens strains

作　　者：鲍长磊 付明哲[1] 何亚鹏[1] 白涛[1] 魏拣选[1] 张彦明[1] 许信刚[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《中国兽医学报》2017年第8期1523-1527,共5页Chinese Journal of Veterinary Science

基　　金：陕西省科技攻关资助项目(2015NY162);西北农林科技大学试验示范站(基地)科技成果推广资助项目(TGZX2015-32)

摘　　要：根据GenBank中已发布的产气荚膜梭菌α,β,ε,ι毒素基因序列,设计并合成4对特异性引物,经过优化多重PCR反应条件,建立了检测产气荚膜梭菌不同毒素型多重PCR方法。特异性试验表明,该方法对A,B,C,D,E型产气荚膜梭菌标准菌株均扩增出了相应的目的条带,而对诺维氏梭菌和腐败梭菌扩增为阴性;灵敏性试验表明,该方法对A,B,C,D,E型标准菌株基因组DNA最低检测量分别为9.0,17.8,12.2,13.8,18.5pg;重复性试验表明,该方法有很好的重复性。应用所建立的方法从21份羊临床病料中检测出9株A型和1株C型产气荚膜梭菌。本试验建立的多重PCR方法可以进行产气荚膜梭菌的快速检测及5种毒素型的鉴别。According to the genome sequences of α,β.ε,t toxins of Clostridium perfringens in Gen- Bank,four pairs of primers targeting α,β.ε,t toxin genes were designed. After the multiplex PCR reaction condition was optimized, the multiplex PCR for identification and toxintyping of C. per- fringens strains was developed. The specificity test showed that the expecled fragments of C. per- fringens reference strains including A,B,C,D,E five toxin types were amplified successfully from genomic DNA of C. perfringens, respectively, ttowever, a hand could not be amplified from Clostidrium novyi and （Tlostridiunz septicum as negative control groups. The sensitivity test showed that the limit detection of multiplex PCR was 9.0,17.8,12.2,13.8,18.5 pg DNA of A,B, C,D,E five toxin types （7. perfringens,respectively. Repetitive testing showed that the established method had a good repeatahility. Nine type A strains of and 1 type C strains of C. Perfringens from 21 clinical samples of dead goat were detected by the muhiplex PCR developed in this study. This study establishes the multiple PCR method which not only can detect C. perfiingens rapidly but also can identify five toxin types of C. perfringens.

关 键 词：产气荚膜梭菌 毒素型 多重PCR 鉴别 

分 类 号：S852.61[农业科学—基础兽医学]