Development Of Quantitative RT-PCR For Detection Of Bovine Rotavirus And Genetic Analysis Of VP6 Gene

Bovine rotavirus(BRV),also known as Calf diarrhea virus,mainly infects calves less than 1 month old,among which calves less than 1 week old are most susceptible to infection.The disease comes on quickly,spreads the scope is wide,forms the large area easily to be popular.For quick accurate diagnosis BRV,this research reference GenBank of bovine rotavirus Sun9 isolates(AB-374146.1),isolates KJ9-1(HM-988974.1),KJ19-2 isolates(MF-940662),KV0418 isolates(EU873011.1),KV0426 isolates(EU873012.1)isolates and M-1(HM235508.1)VP6 full genetic sequence,on the VP6 gene conserved sequence design a pair of specific primers and a probe,to establish a quantitative rt-pcr method for detection of BRV.In addition,a pair of bovine rotavirus VP6 gene-specific primers were designed,and the whole VP6 gene was cloned,the sequence was determined and the genetic analysis was carried out.Through the genetic analysis,the local epidemic strains could be found.The results showed that the best primer concentration was 400 nmol/L,the probe concentration was 350 nmol/L,and the annealing and extension temperature were 60?.The lowest concentration of detected sample is 10copies/?L,which can effectively distinguish bovine viral diarrhea virus,bovine Escherichia coli,bovine Salmonella,bovine infectious rhinotracheitis virus and bovine respiratory syncytial virus.The coefficient of variation of Ct value in and between groups was less than 2%.The linear relationship of the established standard curve was good.The correlation coefficient(R2)was 0.995,and the amplification efficiency(E)was 93.5%.This method has the characteristics of high sensitivity,high specificity and stability.It can be used for the diagnosis and molecular epidemiological investigation of BRV.It provides a reliable method for the prevention and control of bovine diarrhea caused by bovine rotavirus.The total length of VP6 gene was 1194 bp.Compared with the nucleotide sequences of VP6 gene of 23 RVA strains at home and abroad,the homology of VP6 gene is 78.8%-96.6%.The phylogenetic tree of nucleotide showed that the native strain of Inner Mongolia(NM-BRV strain)is closely related to group A rotavirus WC3 strain of the United States,which is the same evolutionary group.