快速检测细菌性腹泻3种厌氧菌多重PCR方法的建立和应用  

Establishment and Application of Multiplex PCR for Rapid Detection of Three Anaerobic Bacteria in Bacterial Diarrhea

作　　者：张凯川 陈柳妃 贾坤 ZHANG Kai-chuan;CHEN Liu-fei;JIA Kun(College of Veterinary Medicine,South China Agricultural University Guangdong Provincial Key Laboratory of Veterinary Clinical Integrated Disease Prevention and Control,Guangzhou 510642,China)

机构地区：[1]华南农业大学兽医学院广东省兽医临床重大疾病综合防控重点实验室,广东广州510642

出　　处：《中国兽医杂志》2022年第2期39-42,47,共5页Chinese Journal of Veterinary Medicine

基　　金：广东省畜禽地方品种保护与开发利用提升工程(2018-143);广东省现代农业产业技术体系(2019KJ127)。

摘　　要：为快速检测厌氧菌引起的细菌性腹泻,建立一种产气荚膜梭菌(Clostridium perfringens)、艰难梭菌(Clostridium difficile)和脆弱拟杆菌(Bacteroides fragilis)的多重PCR检测方法,本试验参照GenBank中产气荚膜梭菌Cpa基因序列、艰难梭菌Glud基因序列和脆弱拟杆菌Leu基因序列上设计合成了3对特异性引物(目的片段分别为326、750 bp和448 bp),通过对PCR反应条件的优化建立多重PCR检测方法。结果显示,多重PCR能从试验的产气荚膜梭菌、艰难梭菌、脆弱拟杆菌扩增出其目的条带,而其他细菌均不能扩增出目的条带;多重PCR对产气荚膜梭菌、艰难梭菌和脆弱拟杆菌的最低检出量分别为1.6×10^(-2)、0.7pg/μL和2.8pg/μL。结果表明本试验建立的多重PCR方法具有良好的稳定性与特异性,为鉴别诊断这3种致病厌氧细菌所导致的腹泻提供一种快速、准确的检测方法。This study aimed to develop a multiplex PCR for rapid detection of Clostridium perfringens,Clostridium difficile and Bacteroides fragilis implicated in bacterial diarrhea.Three pairs of primers were firstly designed from the Cpa gene of C.perfringens,the Glud gene of C.difficile and the Leu gene of B.fragilis for specific amplification of 326,750 bp,and 448 bp products,respectively;and the parameters of this multiplex PCR were then optimized.The results showed that the multiplex PCR allowed highly specific and sensitive amplification of target bands from C.perfringens,C.difficile,and B.fragilis,but not from other bacteria,and had minimum detection amounts of 1.6×10^(-2),0.7 pg/μL,and 2.8 pg/μL for C.perfringens,C.difficile,and B.fragilis,respectively.Thus,the newly established multiplex PCR provides a fast and convenient technique for the detection of three diarrhea-related anaerobic bacterial pathogens.

关 键 词：产气荚膜梭菌 脆弱拟杆菌 艰难梭菌 多重PCR 细菌性腹泻 

分 类 号：S854[农业科学—临床兽医学]