Establishment And Application Of Duplex Real-time PCR Assay For Detection Of CDV And CPV

Canine distemper is an acute and violent infectious disease caused by canine distemper virus (CDV). Canine parvovirus disease is also an acute and violent infectious disease caused by canine parvovirus (CPV). These two major epidemic diseases are badly harmful to the health of dogs and cats. As both of the diseases have similar clinical symptoms, clinical signs and necropsy lesions are not enough for diagnosis. Currently available detection is difficult to meet the clinical needs, so it is urgent to establish a rapid and efficient detection method to detect CDV/CPV, accurately and in large quantities.According to H gene of CDV and sequence VP2of CPV from the GenBank, this study selected the gene sequences of highly conserved and type-specific to devise CDV/CPV specific primers and TaqMan MGB probes. Through matrix methods to screen the best combination of primers and probe concentration and optimize optimum enzyme concentration and annealing temperature, thus establish the detection method of CDV, CPV duplex real-time fluorescence quantitative PCR (fluorescent quantitative real-time PCR, FQ-PCR). The sensitivity, susceptibility, specificity, interference, repetition and compliance of the FQ-PCR method were examined, and clinic suspicious samples infected with CDV/CPV were detected by the FQ-PCR method in contrast to the conventional CDV RT-PCR method and PCR method.The results show that the optimal primer concentration of CDV and CPV with optimized duplex FQ-PCR is0.8μmol/L, the optimal probe concentration of CDV is0.6μmol/L, the optimal probe concentration, enzyme concentration and annealing temperature of CPV is0.7μmol/L,0.05U/μL and60℃respectively. The lowest detection limit of the duplex FQ-PCR method detecting both viruses is1×101copies/μL, and the sensitivity of which is100times more than that of the conventional PCR method.the two standard curve correlation coefficient (R2) is0.997and0.993. The result of the duplex FQ-PCR amplification using the CDV/CPV as the template, is a single specific amplification curve, suggesting that the primers and probe have good specificity. Using the double FQ-PCR to test67known CDV/CPV positive samples, the results prove to be positive. Five kinds of pathogens as control samples and92faeces samples of healthy dogs without CDV/CPV infection were identified using the double FQ-PCR, the results prove negative, indicating that the specificity of the method are high. The results of interference test show that neither of the definite quantity of the two viruses can be affected whether they coexist or not, or there is a large difference in concentration. The result of repeatability test shows that three tests of equal mixture of recombinant plasmids with the concentration of1.0×103~1.0×101copies/μL prove positive while the other three tests with the concentration of1.0×100copy/μL prove negative, indicating that the stability and repeatability of the CDV/CPV duplex FQ-PCR method are good. Forty-eight copies of clinic suspicious samples infected with CDV/CPV were examined using the duplex FQ-PCR method and conventional RT-PCR method, the results show that there have14single positive CDV samples,20single CPV positive samples and4double-positive samples by FQ-PCR.7single positive CDV samples,19single positive CPV samples and2double-positive samples were detected by conventional RT-PCR. It shows that sensitivity of the double FQ-PCR method is higher than the conventional RT-PCR or PCR method.The results demonstrate that, the sensitivity, specificity and stability of FQ-PCR method are very high, and it is suitable for detection of early viruse infection, during, which the quantitation is low. The detection rate of FQ-PCR method is significantly higher than that of conventional PCR method. Thus we can conclude that the double FQ-PCR identification method has the advantage of sensitive, specific, susceptible, rapid, accurate quantification and so on, so it can apply to the clinical detection of CDV/CPV.