牛病毒性腹泻病毒鉴定及分型RT-PCR检测方法的建立  

Establishment of a RT-PCR Assay for Detection and Distinguishing the Genotypes of Bovine Viral Diarrhea Virus

作　　者：李新培[1,2] 朱洪伟 周伟光[2] 关平原 程世鹏 温永俊[1,2] LI Xin-pei;ZHU Hong-wei;ZHOU Wei-guang;GUAN Pin-yuane;CHENG Shi-peng;WEN Yong-jun(State Key Laboratory fbr Molecular Biology of Special Economic Animals,Institute of Wild Econom ic Animals and Chinese Academy of Agricultural Sciences,Changchun,Jilin,ta01 I2,China;Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease,Mini.~try of Agriculture,College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot,Inner Mongolia,010018,China)

机构地区：[1]中国农业科学院特产研究所特种经济动物分子生物学国家重点实验室,吉林长春130112 [2]内蒙古农业大学兽医学院/农业部动物疾病临床诊疗技术重点实验室,内蒙古呼和浩特010018

出　　处：《动物医学进展》2018年第10期6-12,共7页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(31572505);公益性行业(农业)科研专项(201303042)

摘　　要：旨在建立一种特异性检测牛病毒性腹泻病毒(BVDV)的RT-PCR方法,并且可以实现在同一体系中对BVDV进行分型。根据GenBank中公布的BVDV基因组序列,针对高度保守的5′UTR区域分别设计鉴定及分型引物,建立牛病毒性腹泻病毒鉴定及分型RT-PCR检测方法。应用鉴定RT-PCR检测方法可从BVDV阳性毒株中扩增出288bp的特异性片段;而分型RT-PCR检测方法可在同一体系中从BVDV1型和2型毒株中扩增出大小分别为316bp和110bp的特异性片段,但对猪瘟病毒、牛副流感3型病毒及牛传染性鼻气管炎病毒等均为阴性,最低可检测出的病毒量分别为1TCID50/mL和10TCID50/mL。临床试验证明,建立的鉴定及分型RT-PCR检测方法具有良好的特异性、敏感性和重复性,具有很高的实用价值,为进一步开展流行病学调查、病毒分离等提供可靠的技术手段。In order to establish a RT-PCR assay which could detect bovine viral diarrhea virus （BVDV） specifically and distinguish the genotypes of BVDV in one reaction,we designed a pair of target-specific primers in the 5＇UTR region of BVDV conserved gene based on the sequences published in GenBank.Furthermore,we set up a method which can distinguish BVDV genotypes in the same circumstance corresponding to the highly conserved regions of the 5＇UTR region successfully.A 288 bp specific fragment was amplified from the BVDV positive strain by RT-PCR.Two fragments of 316 bp and 110 bp were simultaneously amplified from BVDV type 1 and type 2 strain.No amplification was detected for CSFV,BPIV-3 ,IBRV and so on.The amount of detectable virus was i TCID50/mL, 10 TCID10/mL, respectively.The methods of detection and distinguishing the genotypes which established in this study have good specificity,sensitivity and repeatability,which have practical value significantly,and can serve as reliable technical means for further epidemiological investigation and virus isolation.

关 键 词：牛病毒性腹泻病毒 鉴定 分型 反转录-聚合酶链反应 

分 类 号：S852.653[农业科学—基础兽医学] Q789[农业科学—兽医学]