犊牛四种腹泻病毒多重RT-PCR检测方法的建立及应用OA

To establish a quadruple PCR detection method for Bovine Rotavirus(BRV),Bovine Coronavirus(BCoV),Bovine Viral Diarrhea Virus(BVDV),and Bovine Nebovirus(BNeV),this study designed specific primers targeting BRV-VP6,BVDV-5'UTR,BCoV-NSP2,and BNeV-DR BP.By optimizing the reaction system and conditions,a quadruplex RT-PCR assay capable of simultaneously detecting BRV,BCoV,BVDV,and BNeV was successfully established and applied to clinical samples collected from various re-gions in Yunnan.Results demonstrated that this method specifically detected the target viruses,while other diarrheal pathogens such as BEV,BKV,and BNoV tested negative,indicating good specificity.The lower limits of detection(LOD)for BRV,BCoV,BVDV,and BNeV were 1.34 × 103 copies/μL,3.86 × 103 copies/μL,and 1.06 × 103 copies/μL,3.32 × 103 copies/μL,respectively.Testing of 322 clinical diarrheal samples revealed detection rates of 40.37％for BRV,13.04％for BCoV,10.87％for BVDV,and 1.24％for BNeV.Co-infection rates were 9.94％for BRV+BCoV,4.66％for BRV+BVDV,and 1.24％for BCoV+BVDV.The concordance rate between the quadruplex RT-PCR assay and single RT-PCR assays was 100％.This study provides a rapid and simple detection method for the diagnosis and epidemiological investigation of bovine viral pathogens.The detection results from clinical samples indicate high infection rates of BRV,BCoV,and BVDV in diarrheic calves,with the presence of co-infections.