摘要
根据GenBank已公布的产气荚膜梭菌和腐败梭菌的α毒素基因序列,分别设计并合成针对2种α毒素基因的特异性引物,通过扩增条件的优化,建立快速鉴别产气荚膜梭菌和腐败梭菌的双重PCR方法。特异性试验显示,产气荚膜梭菌和腐败梭菌参考菌株均扩增出了相应的预期目的条带,而大肠埃希菌、沙门菌、金黄色葡萄球菌和链球菌则均未能扩增出相应条带。灵敏度试验结果显示,产气荚膜梭菌和腐败梭菌基因组DNA最低检测量分别为17.95pg/μL和1.261pg/μL。应用该方法对样品进行检测,其中5份为产气荚膜梭菌阳性。成功建立了特异性强、敏感性高的双重PCR方法,可以有效进行产气荚膜梭菌和腐败梭菌的快速鉴别,对羊梭菌病病原快速鉴定及流行病学调查具有重要意义。
According to the sequences published in GenBank,two pairs of specific primers were designed for the specific gene sequences of alpha toxin from C.perfringens and C.septium .A rapid distinguishing PCR for Clostridium perfringens and Clostridium septium was established by optimization of amplification conditions.Specificity test showed the strains of C.perfringens and C.septium both amplified the corresponding expected bands,while no amplifications were detected from Escherichia coli,Salmonella,Staphylococcus aureus and Streptococcus .The sensitivity analysis showed minimum detection amounts of C.perfringens genomic DNA and C.septium genomic DNA were 17.95 pg/μL and 1.261 pg/μL,respectively.Applying this method to detecting samples,the results showed that 5 of them were positive for C.perfringens . This study successfully established a duplex PCR method with high specificity and high sensitivity,which can effectively distinguish C.perfringens and C.septium .And it has a great significance for rapidly distinguishing and epidemiological investigation of the pathogens of clostridial diseases.
作者
李田美
吴正双
许大伟
姜艳芬
LI Tian-mei;WU Zheng-shuang;XU Da-wei;JIANG Yan-fen(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi,712100,China;Animal Supervision Institute of Inner Mongolia Autonomous Region,Huhhot,Inner Mongolia,010051,China)
出处
《动物医学进展》
北大核心
2019年第7期20-25,共6页
Progress In Veterinary Medicine
基金
国家重点研发计划项目(2018YFD0500504)
陕西省农业科技创新与攻关项目(2015NY171)
关键词
产气荚膜梭菌
腐败梭菌
Α毒素
鉴别
双重PCR
Clostridium perfringens
Clostridium septium
alpha toxin
identification
duplex PCR
