副结核分支杆菌实时荧光定量PCR检测方法的建立及应用OA北大核心CSTPCD

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.