BVDV、IBRV和AKAV多重PCR检测方法的建立与应用  

Establishment and application of multiplex PCR assays for BVDV,IBRV and AKAV

作　　者：鲍显伟 李昊 李小龙[1] 石亚楠 王雪妍 许立华[1] BAO Xianwei;LI Hao;LI Xiaolong;SHI Yanan;WANG Xueyan;XU Lihua(College of Animal Science and Technology,Ningxia University,Yinchuan 750021,China)

机构地区：[1]宁夏大学动物科技学院,宁夏银川750021

出　　处：《中国兽医学报》2024年第1期39-43,87,共6页Chinese Journal of Veterinary Science

基　　金：东西部科技合作项目"优质肉牛精准化养殖技术研究与示范"课题(2017BY078)—肉牛健康养殖技术集成与示范;宁夏奶牛育种专项“优质高产奶牛安全健康养殖技术研究与示范”课题(2019NYYZ05)。

摘　　要：牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)、牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)和阿卡斑病毒(Akabane virus,AKAV)是引起牛发生繁殖障碍性传染病的主要病原。本研究根据BVDV的5′-UTR、IBRV的gB基因和AKAV的S基因设计了3对特异性引物,构建了检测BVDV、IBRV和AKAV的多重PCR方法,并优化反应体系和条件,验证了该方法的特异性和敏感性。结果显示,构建的多重PCR方法对其他牛源病毒无扩增,对BVDV、IBRV和AKAV的最低检测限分别为10^(3)、10^(3)和10^(4)拷贝/μL。对采集的184份临床血清样品进行检测,BVDV、IBRV和AKAV的阳性率分别为18.48%、14.13%和1.63%,BVDV和IBRV 2种病原混合感染的阳性率为3.8%,BVDV和AKAV两种病原混合感染的阳性率为1.63%。该结果与我国出入境检疫行业标准符合率达92%以上,BVDV+IBRV和BVDV+AKAV混合感染的符合率为100%。表明本研究建立的多重PCR检测方法可用于临床样品的检测。BVDV、IBRV和AKAV多重PCR检测方法的建立,为我国牛群BVD、IBR和AKA的检测和流行病学调查提供了一种快捷简便的分子生物学检测方法。Bovine viral diarrhea virus(BVDV),infectious bovine rhinotracheitis virus(IBRV)and Akabane virus(AKAV)are the main pathogens that cause reproductive disorders in cattle.In this study,a method was developed for the detection of IBRV and AKAV which are the main pathogens causing reproductive disorders in cattle.Based on the sequences of the 5′-UTR of BVDV,the gB gene of IBRV,and the S gene of AKAV,three specific primer pairs were designed and use to develop the multiplex PCR assay for BVDV,IBRV and AKAV.The results showed that the developed multiplex PCR method did not amplify other viruses of bovine origin,and the minimum detection limits for BVDV,IBRV and AKAV were 10^(3),10^(3) and 10^(4) copies/μL,respectively.184 clinical serum samples were examined for BVDV,IBRV,and AKAV.The positive rates of BVDV,IBRV and AKAV were 18.48%,14.13%and 1.63%,respectively.The positive rates of mixed infections of BVDV and IBRV were 3.8%and 1.63%for BVDV and AKAV.These results were consistent with the entry-exit quarantine industry standard in China with a rate of more than 92%,and the rate of mixed infection of BVDV+IBRV and BVDV+AKAV was 100%.The establishment of the multiplex PCR assay for BVDV,IBRV and AKAV provides a rapid and simple molecular biological method for the detection and epidemiological investigation on BVD,IBR and AKA in cattle.

关 键 词：牛病毒性腹泻病毒 牛传染性鼻气管炎病毒 阿卡斑病毒 多重PCR 

分 类 号：S852.65[农业科学—基础兽医学]