羊源产气荚膜梭菌多重荧光定量PCR检测方法的建立及初步应用  

Establishment and preliminary application of multiplex fluorescent quantitative PCR for detection of Clostridium perfringens from sheep and goats

作　　者：李一鸣 李丽[1,2] 张凯悦 张彦明 姜艳芬[1] LI Yiming;LI Li;ZHANG Kaiyue;ZHANG Yanming;JIANG Yanfen(College of Veterinary Medicine,Northxvest A&F University,Yangling,Shaanxi 712100,China;Weihai Wendeng District Talent Innovation and Development Center,Weihai,Shandong 264400,China)

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]威海市文登区人才创新发展中心,山东威海264400

出　　处：《中国兽医学报》2021年第8期1532-1538,共7页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2018YFD0500500);陕西省农业科技创新与攻关资助项目(2015NY171)。

摘　　要：为了对羊源产气荚膜梭菌(Clostridium perfringens)进行快速检测并定量分型,根据GenBank中产气荚膜梭菌α、β、ε毒素基因序列,分别设计cpa、cpb和etx毒素基因的特异性引物及TaqMan探针,建立羊源产气荚膜梭菌A、B、C、D型多重荧光定量PCR检测方法。结果显示,该方法具有良好的敏感性、特异性和重复性。cpa-pMD18-T、cpb-pGEM-T和etx-pGEM-T质粒拷贝数检测限均可达1×10^(2)拷贝/μL(3.1×10^(-7)mg/L),B型菌液检测限为5.0×10^(2) CFU/mL,基因组DNA检测限为100μg/L;链球菌、沙门菌和大肠埃希菌等在相同条件下扩增均无扩增曲线;批内与批间3次重复试验,Ct值的CV值均小于4%。应用该方法对20份健康山羊粪便样品进行产气荚膜梭菌检测,结果显示共16份阳性样品,其中2份样品同时检测到A和D型菌,其他14份均为A型菌,样品含菌量为10^(2)~10^(4) CFU/g。结果表明,本试验建立的多重荧光定量PCR检测方法不仅为产气荚膜梭菌引发疾病的生物安全提供快速便捷的技术支持,也为这些疾病的临床检测提供辅助手段。In order to quickly detect and quantitatively classify Clostridium perfringens(C.perfringens)from sheep and goats,the specific primers and TaqMan probes of toxin genes cpa,cpb and etx were designed based on the gene sequence ofα,β,εtoxin of C.perfringens,and the multiplex fluorescent quantitative PCR detection method of type A,B,C,D of C.perfringens from sheep and goats was established.The results showed that this method had good sensitivity,specificity and repeatability.The detection limit of cpa-pMD18-T,cpb-pGEM-T and etx-pGEM-T plasmid was up to 1×10^(2) copies/μL(3.1×10^(-7) mg/L),the detection limit of type B bacteria culture and genomic DNA was 5.0×10^(2) CFU/mL and 100μg/L,respectively;Streptococcus,Salmonella and Escherichia coli did not show any amplification curve under the same conditions;CV values of cycle threshold(Ct)were all less than 4%in three repeated tests within and between batches.Then,the method was applied to detect C.perfringens in fece samples from 20 healthy goats.It showed that there were 16 positive samples,among which two samples were detected to have both type A and D C.perfringens,and the other 14 samples only had type A C.perfringens,the content was 10^(2)-10^(4) CFU/g.The multiplex fluorescent quantitative PCR method established in this study not only provided fast and convenient technical support for the biosafety of diseases caused by C.perfringens,but also provided an auxiliary means for the clinical detection of these diseases.

关 键 词：羊源 产气荚膜梭菌 TAQMAN 荧光定量PCR 

分 类 号：S852.61[农业科学—基础兽医学]