Establishment Four Canine Virus TaqMan Real-time QPCR Methods

Canine distemper virus(CDV),canine parainfluenza virus(CPIV),canine parvovirus(CPV),and canine adenovirus type II(CAV-2)are the main pathogens that cause common infectious diseases in dogs,Serious economic losses.In this study,four primers and four TaqMan probes were designed based on the sequences of four viruses registered in GenBank.By optimizing the reaction conditions and sensitivity,specificity,and reproducibility tests,four virus single-factor TaqMan real-time qPCR methods and quadruple TaqMan real-time qPCR method.The results showed that the four virus single-plex methods had a minimum detection volume of 101copies/?L for CAV-?,CPVand CPIV,and a minimum detection volume of CDV of 102-101 copies/?L,with high sensitivity;linearity of the standard curve Good,the correlation coefficient(R2)is greater than 0.990;the four viruses have amplification signals only in the positive template,which has strong specificity.The coefficients of variation within and between the two groups of CPV and CAV-? viruses were less than 2%,and CPIV and CDV viruses were less than 3%,with good repeatability.Tested 72 samples,all four viruses were specifically detected.The sensitivity of the quadruple method,CAV-? and CDV can reach 102 copies/?L,CPV and CPIV can reach 103 copies/?L,the sensitivity is higher;the standard curve has a good linear relationship and the correlation coefficients(R2)are all greater than 0.990.There is no cross-reaction between different viruses,and only the positive template has an amplification signal,which has strong specificity.The established method has good repeatability,and the coefficients of variation within and between groups are less than 5%.Tested 26 samples,The results were consistent with the single-check method.It shows that the established quadruple TaqMan real-time qPCR detection method has the advantages of fast,sensitive and stable,and can be used for the quantitative detection of four viruses.