犬冠状病毒、犬细小病毒TaqMan荧光定量PCR检测方法的建立及应用OA

This study aimed to establish a TaqMan real-time PCR assay for the detection of canine coronavirus(CCoV)and canine parvovirus(CPV).Specific primers and TaqMan probes were designed targeting the M gene of CCoV and the VP2 gene of CPV.The primer and probe concentrations,reaction system,and amplification conditions were optimized,and the sensitivity,specificity,and reproducibility of the assay were evaluated.The results showed that for the positive reference plasmids of CCoV and CPV,within the concentration range of 10 to 108 copies/μL,the correlation coefficients(R2)were 0.999 and 0.997,respectively.Both real-time PCR assays exhibited good linearity,with a minimum detection limit of 10 copies/μL.Specificity tests revealed no amplification curves for canine adenovirus type 2,canine parainfluenza virus,canine distemper virus,or rabies virus.In reproducibility tests,the intra-assay and inter-assay coefficients of variation were less than 2％.Testing of 78 clinical samples showed that the detection rate of this method for CPV and CCoV was improved compared to conventional PCR.In conclusion,the established TaqMan real-time PCR assay is characterized by high sensitivity and specificity,making it suitable for clinical detection and epidemiological investigation of CCoV and CPV.