Establishment And Application Of Detection Method For BVDV Genotyping

Bovine viral diarrhea virus(BVDV)is one of the most important pathogens causing great economic losses in cattle industry worldwide.To quickly detect BVDV and distinguish between different genotypes of BVDV,according to the test in GenBank BVDV three genotypes of the translation section 5’end,design specific primers and probe respectively,through the optimization of primers and probe and the annealing temperature,establish BVDV genotyping triple rt-pcr method,BVDV genotyping triple nano rt-pcr method and BVDV genotyping triple fluorescent quantitative rt-pcr method,and the Hohhot area to zap the nose swabs and fecal samples of molecular epidemiology investigation.The results showed that the three methods of BVDV genotyping could only detect BVDV-1,BVDV-2 and BVDV-3 nucleic acids from various pathogens causing diarrhea and respiratory diseases in bovine,which indicated that all the three methods had good specificity.The lowest nucleic acid levels of BVDV-1,BVDV-2 and BVDV-3 were 1×102 copies/μL,1×102 copies/μL and 1×103 copies/μL,respectively,among the three diagnostic methods of BVDV genotyping.The minimum nucleic acid content of BVDV-1,BVDV-2 and BVDV-3 was 1×102 copies/μL,while the minimum nucleic acid content of BVDV-1,BVDV-2 and BVDV-3 was 1×101copies/μL,1×102 copies/μL and 1×101copies/μL,respectively,indicating the high sensitivity of the three methods.Among the three diagnostic methods,the target fragment with the same size as the target band was repeatedly amplified by common RT-PCR and nano-PCR,and the intra-group and inter-group coefficient of variation of fluorescent quantitative RT-PCR were less than 2%,indicating that all the three diagnostic methods had good reproducibility.At the same time,238 samples of calves and dairy cows in Hohhot were detected by triple fluorescence quantitative RT-PCR,and the 5’UTR of some positive samples of BVDV was analyzed.The results showed that 48 cases were positive(detection rate was 20.16%).Among them,46 cases were positive for BVDV-1 and 2 cases were positive for BVDV-2.The most prevalent subtypes of BVDV-1 in Hohhot were BVDV-1a,BVDV-1b and BVDV-1c.The two detected BVDV-2 subtypes are BVDV-2a.This study provides different means for the rapid detection of BVDV and different genotypes,and can be used for the molecular epidemiological investigation of BVDV and the rapid diagnosis of bovine viral diarrhea.The epidemiological survey results can provide a reference for the formulation of comprehensive prevention and control measures.