动物医学进展2024，Vol.45Issue(7)：1-6,6.
副结核分支杆菌实时荧光定量PCR检测方法的建立及应用
Development and Application of a Real-time Quantitative PCR Assay for Detection of Mycobactium paratuberculosis
摘要
Abstract
In order to enhance the diagnosis,monitoring,prevention and control of Mycobacterium paratuberculosis[Mycobacterium avium subsp.paratuberculosis(MAP)],a novel real-time quantitative PCR detection method was developed.In this method,a pair of precise primers and Taq Man probe were designed targeting the specific insertion sequence IS900 of MAP epidemic strains in China,and a recombinant positive plasmid serves as the template for es-tablishing the qPCR methodology.The optimized reaction conditions ensure specificity,sensitivity and repeatability of the method.The method achieves a minimum detection limit of 5 copies/μL,with a reproducibility coefficient of variation under 4％.Importantly,there is no cross-reaction in the detection of IBRV,BPIV-3,BRV,BVDV,Myco-plasma bovis,Klebsiella pneumoniae,Mannkimia,and MAP could be specifically detected.Applied to clinical sam-ples from 6 cities of Inner Mongolia Autonomous Region,the method detects an average MAP positive rate of 0.85％(6/704).This successful qPCR method offers a swift tool for diagnosis and surveillance,which can be used for the monitoring and investigation of MAP in clinical practice.关键词
副结核分支杆菌/实时荧光定量PCR/检测方法/感染调查Key words
Mycobactium paratuberculosis(Mycobacterium avium subsp.paratuberculosis)/real-time fluo-rescence quantitative PCR/detection method/infection investigation分类
农业科技引用本文复制引用
平宇明,常华,李劼,张宏莉,班亚星,刘建奇,任希恩,邬彩丽,刘东霞,徐丽媛,杨雪娇..副结核分支杆菌实时荧光定量PCR检测方法的建立及应用[J].动物医学进展,2024,45(7):1-6,6.基金项目
呼和浩特市科技创新领域人才项目(2022RC-联合体-1) （2022RC-联合体-1）
云南省基础研究面上项目(202001AT070119) （202001AT070119）
2023年生物医药领域科技计划项目"抗畜禽腹泻兽药创制"子项目(K2420230005) （K2420230005）
云南省农业基础研究联合专项面上项目(2018FG001-043) （2018FG001-043）
云南农业大学兽医公共卫生省创新团队项目(202105AE160014) （202105AE160014）
云南省石林县奶山羊产业科技特派团项目(202104BI090017) （202104BI090017）
