Establishment Of The Real-time Fluorescence RT-PCR For Bovine Parainfluenza Virus 3

This study was designed to establish a real-time fluorescence quantitative PCR which is fast, accurate, strong specificity and high sensitivity to identify BPIV3. According to the complete sequences of BPIV3 published in GenBank, the sequences were comparatively analyzed and a pairs of primers and TaqMan probes were designed and composed which can amplify BPIV3 specifically. Standard positive plasmid DNA templates were constructed. The reaction parameters and conditions of the 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 real-time fluorescence quantitative PCR reaction system established in this study and use this method to detect 11 clinic samples. The results showed that to reach the best reaction effect in this 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 BPIV3 specifically. Also it can differentiate with pasteurella,streptococcus,sheep pox virus, pox virus, orf virus, peste des petits ruminants virus,IBR,mycoplasma infectious bovine rhinotracheitis virus. In addition, the coefficient of variation of this method Ct values are less than 0.1. The minimum detectable count of BPIV3 were 1.00 x 102 copies/ L respectively. The results indicated that this established 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 BPIV3.