产气荚膜梭菌菌落多重PCR方法的建立及初步应用

根据GenBank中已发布的产气荚膜梭菌α、β、ε、τ毒素基因序列，分别设计并合成针对4种毒素基因的特异引物，通过优化多重PCR反应条件，建立1种简单的产气荚膜梭菌定型菌落多重PCR方法。结果显示：A、B、C、D、E5型产气荚膜梭菌参考菌株均扩增出了相应的预期目的条带，而大肠杆菌、巴氏杆菌和芽孢杆菌则均未能扩增出相应条带；将单个菌落稀释100倍，仍能扩增出相应的目的片段，该方法对B型和E型参考菌株最低检测量分别为2．6×10^4cfu／mL、1．2×10^4cfu／mL。应用该多重PCR方法从106份样品中检测到30株产气荚膜梭菌且均为A型，其中病死鸡的盲肠内容物分离率为36．5％（19／52），健康鸡群新鲜粪便样品分离率为20．4％（11／54）。本研究建立的多重PCR方法特异性强，敏感度高，重复性好，可以有效进行产气荚膜梭菌的快速检测及5种血清型的鉴别，对产气荚膜梭菌的感染及食品安全问题的研究均具有重要意义。

Development and preliminary application of colony multiplex PCR for serotyping of clostridium per f ringens strains

According to the highly conserved genome sequences of α、β、ε、τ toxins of Clostridium perfringens,four pairs of primers targeting α、β、ε、τ toxin genes were designed. The multiplex PCR reaction condition was optimized and the colony multiplex PCR of identification and serotyp- ing of C. perfringens strains were established. The expected bands of C. perfringens reference strains including A,B,C,D,E 5 serotypes were all amplified successfully by the colony multiplex PCR assay, respectively. However, the bands could not be amplified from E. coli, Pasteurella and Bacillus. The expected bands were also amplified from C. perfringens single colony when diluted to 100 times,as well as from the diluted fresh cultures of the reference strains when type 13 was 2.6 ×10^ cfu per mL and type E was 1.2 ×10^ cfu per mL,respectively. C. perfringens was isolated from 19 of 52 cecal contents samples of dead chicken （36. 5%） and 11 of 54 fecal samples of healthy chicken（20.4%）,those isolates were identified and confirmed as type A C. perfringens by the multiplex PCR developed in this study. The established multiplex PCR is specific, sensitive and repeatable,and can rapidly detect and serotype C. perfringens effectively. The method laid a basis for the future study on C. perfringens infection in animal and food safety issue.