摘要
为建立一种可以快速检测牛冠状病毒(BCoV)、牛肠病毒(BEV)、牛病毒性腹泻病毒(BVDV)的实时荧光PCR方法并应用于临床检测,根据BCoV N基因、BEV 5'-UTR基因、BVDV 5'-UTR基因设计3对特异性引物和探针,通过对引物、探针的浓度以及反应条件的优化,建立了牛冠状病毒、牛肠病毒、牛病毒性腹泻病毒三重一步法实时荧光PCR方法,并对该三重PCR方法的敏感性、重复性、特异性和临床样本检测结果进行了评价.结果显示,该方法灵敏度高,对BCoV、BEV和BVDV的最低检测限为10 copies/pL;重复性稳定,批内和批间变异系数(CV)均在2％以下;特异性强,对常见腹泻类病原无交叉反应.应用此方法检测河南省445份牛腹泻粪便样本,BCoV、BEV和BVDV的阳性率分别为6.5％(29/445)、17.5％(78/445)、12.1％(54/445).上述结果表明,本研究建立了牛冠状病毒、牛肠病毒、牛病毒性腹泻病毒一步法三重实时荧光PCR方法,为疫病的快速检测及预防提供了有效的技术手段.
Abstract
To establish a rapid real-time fluorescence PCR method for the detection of bovine coro-navirus(BCoV),bovine enterovirus(BEV),and bovine viral diarrhea virus(BVDV)in clinical diagnostics,three sets of specific primers and probes were designed based on the BCoV N gene,BEV 5'-UTR gene,and BVDV 5'-UTR gene.Through optimization of primer and probe concentrations,as well as reaction condi-tions,a one-step triplex real-time fluorescence PCR method was developed for simultaneous detection of bovine coronavirus,bovine enterovirus,and bovine viral diarrhea virus.The sensitivity,repro-ducibility,specificity,and clinical sample detection results of this triplex PCR method were as-sessed.The results demonstrated high sensitivity,with the minimum detection limits for BCoV,BEV,and BVDV being 10 copies/μL.Reproducibility was stable,with intra-batch and inter-batch coefficients of variation(CV)both below 2％.The method exhi-bited strong specificity,showing no cross-reaction with common diarrheal pathogens.This method detected positive rates of 6.5％(29/445)、17.5％(78/445)and 12.1％(54/445),respectively,in 445 bovine diarrhea fecal samples from Henan Province.These findings indicate the successful establishment of a one-step triplex real-time fluorescence PCR method for BCoV,BEV,and BVD-V,providing an effective technical approach for the rapid detection and prevention of these diseases.
基金项目
河南省科技重大专项(2022)(221100110600)
河南省重点研发与推广专项(2023)(232102111053)
国家科技期刊平台
NSTL
万方数据