Establishment Of The Real-time Fluorescence Quantitative PCR For Detection Method Of Capripoxvirus

One pair of specific primer and two TaqMan probes were designed and synthesized according to nucleotide sequence of A29L gene of goat pox virus (AY077836) and sheep pox virus (AY077832) in GenBank.And Standard recombinant plasmid were constructed. The real time fluorescent quantitation PCR method of detecting Capripoxvirus was establish by selecting and optimizing annealing temperature, concentration of primers and the probe concentration and the standard positive plasmid templates of Capripoxvirus were done ten-fold serial dilutions to make the real-time fluorescence quantitative PCR standard curve. The sensitivity, specificity and reproducibility of the establishing real-time fluorescence quantitative PCR method was assessed,and clinical samples was detected by this method.The results showed the real-time fluorescent quantitation PCR reaction condition are that the annealing temperature is 58.4℃,the concentration of primer is 400 nmol/L, and the concentration of probe is 200 nmol/L. The detection method has no cross-reaction with other four kinds of virus, and goat pox virus and sheep pox virus also has no cross-reaction. The coefficient of variation of repeatability test of the method are less than 2%, and the dual real-time fluorescent quantitative PCR detection limit of lowest concentration was 470 fg and 440 fg respectively. The correlation coefficient of standard curve was 0.995 and 0.997 respectively by quantitative tests of standard template. The result shows that the method is in good specificity, sensitivity and stability, which can be used to accurately conduct a quantitative determination on GTPV and SPPV.19 clinical samples was detected, GTPV positive has 14,SPPV positive has 4, SPPV and GTPV mixed infection has 1.