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方法的建立与应用  

Establishment and application of a one-step multiple fluorescence quantitative PCR method for detection of BCoV,BEV and BVDV

作　　者：康台生 刘影 胡煜锋 李博文 王益冰 王淑娟[2] 闫若潜[2] 王东方[2] KANG Taisheng;LIU Ying;HU Yufeng;LI Bowen;WANG Yibing;WANG Shujuan;YAN Ruoqian;WANG Dongfang(College of Animal Science and Technology,Henan University of Science and Technology,Luoyang 471023,China;Henan Center for Animal Diseases Control&Prevention/Henan Provincial Key Laboratory of Monitoring,Early Warning and Prevention and Control of Major Animal Diseases,Zhengzhou 450008,China;College of Animal Science and Veterinary Medicine,Henan A gricultural University,Zhengzhou 450002,China)

机构地区：[1]河南科技大学动物科技学院,河南洛阳471023 [2]河南省动物疫病预防控制中心/河南省重大动物疫病监测预警及防控重点实验室,河南郑州450008 [3]河南农业大学动物医学院,河南郑州450002

出　　处：《中国兽医科学》2024年第4期472-478,共7页Chinese Veterinary Science

基　　金：2022年度河南省重大科技专项(221100110600);2023年度河南省重点研发与推广专项(232102111053)。

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/μL;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。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 thesediseases.

关 键 词：牛冠状病毒 牛肠病毒 牛病毒性腹泻病毒 一步法PCR 检测 

分 类 号：S852.659.6[农业科学—基础兽医学]