Establishment And Application Of Multiple Fluorescence Quantitative PCR Method For Bovine Viral Diarrhea Virus And Bovine Rotavirus

With the tendency of intensive and large-scale breeding of cattle industry,the mass diarrhea has become more and more harmful,and it is necessary to prevent and control the disease.The reasons are complex that can cause cattle diarrhea but viral diarrhea is the most serious and common,consisting bovine viral diarrhea virus(BVDV)and bovine rotavirus(BRV).BVDV is widely distributed around the world.Infected animals can cause symptoms in the digestive tract,respiratory tract,and reproductive tract,as well as continuous detoxification and immunosuppression.BRV is endemic and severely restricts the growth and development of the herd,high incidence of 90%,treatment delay can lead to the productivity descend.Co-infection of BVDV and BRV caused obstacles of pathogen diagnosis.In this study,multiplex taqman fluorescence quantitative PCR method was established to detect three viruses of BVDV type 1、BVDV type2 and BRV,which applied to detect clinical samples and epidemiological investigation.And the evolution graphs of the virus can be drawn though genetic analysis.The study provide reference for the prevention and control of viral pathogens in my country.1 Establishment of multiple fluorescence quantitative PCR detection method for BVDV-1,BVDV-2 and BRVBovine viral diarrhea virus(BVDV)and bovine rotavirus(BRV)infections cause cattle mass diarrhea,and the differential diagnosis is of great significance.Three pairs of specific primers and three probes were designed for the 5’UTR gene of bovine viral diarrhea virus type1and type 2,the VP6 gene of bovine rotavirus,a multiplex taqman q RT-PCR method capable of detecting three viruses simultaneously was established and its standard curve,specificity,sensitivity and reproducibility were tested.The results showed that the method had a good linear relationship and amplification efficiency;the high specificity show it amplified a typical amplification curve for the target virus,no cross-reaction between different viruses;the sensitivity result show that the detection limit respectively was BVDV-1 1×10~1 copies/μL,BVDV-2 1×10~1 copies/μL,BRV 1×10~2 copies/μL,the sensitivity was at least 10 times higher than that of conventional PCR;the repeatability was good because the coefficient of variation was less than 3%.100 clinical samples were tested using the method and technical specification and reported method,the coincidence rates of BVDV and BRV are 91.3%and 96%,respectively.This method can be used for rapid clinical testing in laboratory.2 Molecular epidemiological investigation of BVDV and BRVTo determine the nationwide prevalence and genetic diversity of bovine viral diarrhea virus(BVDV)and bovine rotavirus(BRV)in our country,60 dairy farms with 980 clinical samples in 15 provinces of China were surveyed by the taqman q PCR in 2019-2021.The results showed that the positive rate of BVDV in different regions was 0%-23.33%.A total samples positive rate was 16.22%(159/980).In addition,32of 60(53.33%)farms were identified as positive by q PCR.Another results show the positive rate of BRV in different regions was 0%-55%,A total samples positive rate was 12.96%(127/980),In addition,26of 60(43.33%)farms were identified as positive by q PCR.The positive rate of BVDV and BRV is different in every province,and the highest is in the northwest.The positive rate of BVDV in Winter and spring is significantly higher than summer and autumn.The positive rate of BRV is not obvious in every season.The study provides foundation for the molecular epidemiological investigation,prevention and control diseases in future.3 BVDV 5’UTR genetic evolution analysisTo determine the nationwide genetic diversity of bovine viral diarrhea virus(BVDV)in our country.In this study,BVDV 5’UTR genetic evolution analysis by some positive samples with BVDV during 2019-2021.A total of 25 sequences were obtained and phylogenetic analysis of these 5’UTR sequences revealed the homology is 70%-100%,the presence of 3 different sub-genotypes of BVDV-1 including 1a(n=16),1c(n=5),1m(n=4),respectively.It is speculated that BVDV-1a,1c,and 1m were the dominant strains in China.The study provides reference for the molecular epidemiological investigation and vaccine research.of the disease in future.