牛病毒性腹泻病毒两种基因型双重RT-PCR检测方法的建立及应用  

Establishment and Application of Duplex RT-PCR for Genotypes of Bovine Viral Diarrhea Virus

作　　者：王勤[1] 何博[1] 欧云文 刘俐君 杨磊 潘琴 张杰[3] WANG Qin;HE Bo;OU Yun-wen;LIU Li-jun;YANG Lei;PAN Qin;ZHANG Jie(Dazhou Vocational and Technical College,Dazhou,Sichuan,635001,China;Animal Disease Prevention and Control Center of Kaijiang County,Dazhou,Sichuan,636250,China;State Key Laboratory of Veterinary Etiological Biology,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou,Gansu,730046,China;Animal Disease Prevention and Control Center of Dazhou,Dazhou ,Sichuan,635000,China)

机构地区：[1]达州职业技术学院,四川达州635001 [2]开江县动物疫病预防控制中心,四川达州636250 [3]中国农业科学院兰州兽医研究所/家畜疫病病原生物学国家重点实验室,甘肃兰州730046 [4]达州市动物疫病预防控制中心,四川达州635000

出　　处：《动物医学进展》2021年第9期24-29,共6页Progress In Veterinary Medicine

基　　金：四川省科技计划资助项目(2019JDRC0110,2020JDRC0146);四川县域经济发展研究中心资助项目(xy2020055);“西部之光”访问学者培训计划联合资助项目。

摘　　要：为建立牛病毒性腹泻病毒(BVDV)双重RT-PCR检测方法,根据BVDV-1(AF091605.1、KF501393.1、MK204904.1、MN513404.1和MG923683.1)和BVDV-2(AF502399.1、MG436782.1、AF145969.1、KC176777.1和MN513411.1)流行毒株5′-UTR保守序列设计2对特异性引物,通过优化反应条件,建立可同时检测BVDV-1和BVDV-2的双重RT-PCR方法。结果显示,该检测方法重复性好、特异性强,可分别检测出BVDV-1和BVDV-2核酸,与牛传染性鼻气管炎病毒(IBRV)、牛轮状病毒(BRV)、牛冠状病毒(BCV)、牛副流感病毒3型(BPIV-3)、猪瘟病毒(CSFV)均不发生交叉反应;最低检测量为1×103拷贝/μL和1×102拷贝/μL;对达州及周边地区的332份临床样品进行检测,结果表明,该地区BVDV-1阳性率为7.83%,BVDV-2阳性率为0.90%,未检出BVDV-1和BVDV-2混合感染的临床样品。综上所述,建立的双重RT-PCR检测方法具有良好的特异性、敏感性、重复性和临床适用性,可用于BVDV的临床诊断和分子流行病学调查。This experiment was aimed to establish duplex RT-PCR for bovine viral diarrhea virus.According to the difference of 5′-untranslated region(5′-UTR)between BVDV-1(GenBank:AF091605.1,KF501393.1,MK204904.1,MN513404.1 and MG923683.1)and BVDV-2(GenBank:AF502399.1,MG436782.1,AF145969.1,KC176777.1 and MN513411.1),two pairs of primers were designed.The reaction conditions were optimized to establish duplex RT-PCR for BVDV-1 and BVDV-2.The test results showed that the duplex RT-PCR can respectively detect genome of BVDV-1 and BVDV-2 with a better repeatability,specificity,and no cross reaction with IBRV,BRV,BCV,BPIV-3 and CSFV.The detection limits of BVDV-1 and BVDV-2 genome were respectively 1×103 copy/μL and 1×102copy/μL.332 clinical samples which collected in Dazhou and surrounding areas were tested.The results suggested that the positive rates of BVDV-1 and BVDV-2 were respectively 7.83%and 0.90%,and there was no clinical samples with mixed infection of BVDV-1 and BVDV-2.In summary,the duplex RT-PCR has good specificity,sensitivity,repeatability and clinical applicability,and can be used for clinical diagnosis and molecular epidemiological investigation of BVDV.

关 键 词：牛病毒性腹泻病毒1型 牛病毒性腹泻病毒2型 双重反转录-聚合酶链反应 

分 类 号：S852659.6[农业科学—基础兽医学] S858.23[农业科学—兽医学]