Establishment And Application Of RT-LAMP Detection Methods For BVDV,BRV And BCoV

Bovine viral diarrhea virus(BVDV),Bovine rotavirus(BRV)and Bovine coronavirus(BCo V)are the three main pathogenic pathogens causing Bovine diarrhea disease.The frequency of mixed infection or bacterial secondary infection is high and the clinical symptoms are not easy to distinguish,which brings great difficulties to the field rapid detection of diarrhea of calves.Therefore,this study aims to establish loop-mediated isothermal amplification(RT-LAMP)for the rapid detection of BVDV,BRV and BCo V,respectively,to provide a simple,sensitive,accurate and reliable tool for the three pathogens.In this study,the specificity of RT-LAMP primers were designed for the conserved regions(BVDV5’UTR,BRV NSP5 and BCo V N gene sequences)according to the reference virus strains of BVDV,BRV and BCo V in Gen Bank.By optimizing the system and reaction conditions,the sensitivity and specificity tests of the method and the stool samples of clinical diarrhea were tested,and three RT-LAMP methods which could specifically amplify BVDV,BRV and BCo V were established.The results showed that the optimal reaction conditions of BVDV rt-lamp were as follows:the sensitivity was 1×10~2 copies/μL at 64℃for 45min,and the sensitivity of ordinary PCR was 1×10~3 copies/μL.The best LAMP reaction conditions were 61℃for40min,and the sensitivity of BRV rt-lamp was 1×10~2copies/μL,and that of conventional PCR was 1×104 copies/μL.The optimal reaction condition of BCo V rt-lamp was 60℃for 40min,and the sensitivity of rt-lamp was 1×10~2 copies/μL and that of ordinary PCR was 1×10~3 copies/μL.Three methods were used to amplify escherichia coli,Salmonella,Bovine parainfluenza virus type 3(BPIV3),bovine infectious rhinotracheitis virus(IBRV),and no positive results were found.The positive rate of BVDV was 78.9%,,the positive rate of ordinary PCR was 58%.The positive rate of BCo V was 18.4%,the positive rate of ordinary PCR was 10.5%,the positive rate of BRV was 28.9%,and the positive rate of ordinary PCR was 15.8%.This test provided an efficient,specific and simple method for clinical detection of BVDV,BRV and BCoV.