Establishment Of The Real-time Fluorescence Double PCR To Identify CSFV And BVDV1

This study was designed to establish a duplex real-time fluorescence quantitative PCR which is fast, accurate, strong specificity and high sensitivity to identify CSFV and BVDV. According to the complete sequences of CSFV and BVDV 1 published in GenBank, the sequences were comparatively analyzed and two pairs of primers and TaqMan probes were designed and composed which can amplify CSFV and BVDV1 specifically. Standard positive plasmid DNA templates were constructed. The reaction parameters and conditions of the duplex real-time fluorescence quantitative PCR were optimized and the real-time fluorescence quantitative PCR standard curve was drew. Assess the sensitivity, specificity and reproducibility of the duplex real-time fluorescence quantitative PCR reaction system established in this study and use this method to detect 86 clinic samples. The results showed that to reach the best reaction effect in this duplex real-time fluorescent quantitation PCR, the concentration of primer and probe were 400 nmol/L and 300 nmol/L respectively, and the annealing temperature was 58℃. This diagnostic method can detect CSFV and BVDV 1 specifically. Also it can differentiate with sheep pox virus, pox virus, orf virus, peste des petits ruminants virus, infectious bovine rhinotracheitis virus. In addition, the coefficient of variation of this method Ct values are less than 0.01. The minimum detectable count of CSFV and BVDV 1 were 1.04×102 copies/L and 1.15× 102 copies/L respectively. The results indicated that this established duplex real-time fluorescence quantitative PCR method had good stability and high sensitivity. This PCR method was applied to detect clinical samples, and it can be fast and accurate to identify CSFV and BVDV1.