Establishment And Application Of Fluorescence Quantitative RT-PCR For Detection Of Bovine Respiratory Syncytial Virus

Bovine respiratory and cytoplasmic viral disease is an infectious disease that caused by cow respiratory and cytoplasmic virus.This disease has become a worldwide epidemic.In order to diagnose the disease quickly and accurately,the conserved sequences of N gene of BRSV were selected.Specific primers and probes were designed and synthesized and to establish fluorescence quantitative RT-PCR detection method for bovine respiratory syncytial virus and then to detect nucleic acid samples of bovine nasal swab.The test results showed that the optimal primers concentration for this method were 400 nmoL/L,the probe concentration was 250 nmoL/L,the annealing and extension temperatures were both 60°C.Moreover,the sensitivity was high,the lowest sample concentration that can be detected was 10 copies/?L,and strong specificity,can effectively distinguish between bovine viral diarrhea virus,bovine rhinotracheitis virus,bovine parainfluenza virus type 3,bovine mycoplasma,Pasteurella type A,and good repeatability,the coefficient of variation of intragroup and intergroup Ct were less than 2%.The established standard curve had a good linear relationship with a correlation coefficient of(R2)? 1.000 and an amplification efficiency of(E)? 92.4%.In addition,33 samples of nasal swabs collected from a cattle farm in Inner Mongolia were subjected to fluorescence quantitative RT-PCR established this study.The results showed that 5 BRSV positive samples were detected in 33 nucleic acid samples at Ct value was 38 or so.Moreover,in order to verify the detected BRSV-positive nucleic acid,the amplified gene fragments were cloned and sequenced.The results showed that the amplified fragments of 5 samples were all 205 bp,and the homology with the reference sequence was 99.0 % and 99.5 %.The experimental results showed that this study established a fluorescence quantitative RT-PCR method for the detection of BRSV,with high sensitivity,strong specificity and stability.It can used to diagnosis and molecular epidemiological investigation of BRSV,and provided a reliable method for prevent and control bovine respiratory syncytial virus disease in cattle breeding industry.