产气荚膜梭菌的分离鉴定及PCR检测方法的建立

发布时间：2018-07-23 09:28

【摘要】：产气荚膜梭菌是引起多种人畜共患病的条件致病菌,为了解内蒙古地区牛、羊产气荚膜梭菌的型的分布情况,根据GenBank上发表的产气荚膜梭菌16S rRNA基因和编码α、β、ε毒素的基因设计了 4对引物,建立建立产气荚膜梭菌16SrRNA的PCR检测方法和产气荚膜梭菌α、β、ε毒素的多重PCR分型方法,并对采集的64份病料进行分离、培养和生化鉴定,然后用上述建立的PCR方法对分离菌进行检测与分型。构建产气荚膜梭菌16S rRNA基因的重组质粒,分析其与产气荚膜梭菌参考菌株的同源性并构建进化树。试验结果表明:建立的产气荚膜梭菌16S rRNA PCR方法和产气荚膜梭菌α、β、ε毒素的多重PCR分型方法均能扩增出相应的条带,并且敏感性和特异性均比较高。对采集的64份病料中的细菌进行分离、培养,并经革兰氏染色镜检,生化鉴定后,有11株分离菌与国内外报道的产气荚膜梭菌的形态及生化特性一致。用上述两种PCR方法对这11份分离菌进行检测,扩增出的条带大小与预期的一致,其中多重PCR结果显示,这11株分离菌中有3株A型产气荚膜梭菌,3株C型产气荚膜梭菌,5株D型产气荚膜梭菌。测序结果表明这11株产气荚膜梭菌的16S rRNA和参考菌株的16S rRNA的同源性在98%以上。
[Abstract]:Clostridium perfringens is a conditional pathogen causing zoonosis. In order to understand the distribution of Clostridium perfringens in Inner Mongolia, Based on the 16s rRNA gene of Clostridium perfringens and the gene encoding 伪, 尾, 蔚 toxin published in GenBank, four pairs of primers were designed to establish the PCR detection method of Clostridium perfringens 16s rRNA and the multiplex PCR typing method of Clostridium perfringens 伪, 尾, 蔚 toxin. 64 samples were isolated, cultured and biochemically identified, and then the isolated bacteria were detected and typed by the PCR method. The recombinant plasmid of 16s rRNA gene of Clostridium perfringens was constructed and its homology with the reference strain of Clostridium perfringens was analyzed and an evolutionary tree was constructed. The results showed that the 16s rRNA PCR method of Clostridium perfringens and the multiplex PCR typing method of Clostridium perfringens 伪, 尾, 蔚 toxin could amplify the corresponding bands, and the sensitivity and specificity were high. The bacteria collected from 64 samples were isolated, cultured and identified by Gram staining. After biochemical identification, 11 isolates were found to be consistent with the morphological and biochemical characteristics of Clostridium perfringens reported at home and abroad. The two PCR methods were used to detect the 11 isolates. The amplified bands were in accordance with the expected size, and the multiplex PCR results showed that, Of the 11 isolates, 3 were Clostridium perfringens type A, 3 were Clostridium perfringens type C and 5 were Clostridium perfringens type D. The results of sequencing showed that the homology of 16s rRNA between the 11 strains of Clostridium perfringens and the reference strain was more than 98%.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

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