非洲猪瘟病毒K196R基因实时荧光定量PCR检测方法的建立  

Establishment of real-time quantitative PCR assay for detection of African swine fever virus K196R gene

作　　者：崔贝贝 仇松寅[1] 梅琳[1] 韩雪清[1] 吴绍强[1] 李霆[1] 林祥梅[1] CUI Bei-bei;QIU Song-yin;MEI Lin;HAN Xue-qing;WU Shao-qiang;LI Ting;LIN Xiang-mei(Chinese Academy of Inspection and Quarantine,Beijing 100176,China)

机构地区：[1]中国检验检疫科学研究院

出　　处：《中国兽医杂志》2019年第5期3-8,共6页Chinese Journal of Veterinary Medicine

基　　金：国家重点研发计划(2017YFD0502305-3)

摘　　要：为建立一种准确、特异、高效、快速的非洲猪瘟病毒定量检测方法,本研究根据非洲猪瘟病毒(African swine fever virus,ASFV)早期表达基因K196R的基因序列,设计了TaqMan荧光定量PCR引物及探针,通过优化退火温度、引物及探针浓度,建立了快速检测ASFV的TaqMan荧光定量PCR检测方法。结果表明,该方法选择的引物具有高度灵敏性和特异性,以构建的重组质粒为标准品建立的TaqMan荧光定量PCR方法的标准曲线具有良好的线性关系(R2=0.998),对ASFV核酸最低检测下限为1.3拷贝,且与猪伪狂犬病病毒、猪细小病毒、猪圆环病毒2型等多种病原不存在交叉反应。本研究建立的K196R基因实时荧光定量PCR检测方法为非洲猪瘟疫情提供了一种新型、灵敏和特异的早期检测方法。In order to establish an accurate,specific,efficient and rapid quantitative detection method for African swine fever virus,this study designed TaqMan real-time PCR primers and probe based on the gene sequence of African swine fever virus(ASFV)early expression gene K196R.By optimizing the annealing temperature,primers and probe concentration,a TaqMan real-time PCR method for rapid detection of ASFV was established.The results showed that the primers selected by this method were highly sensitive and specific.The standard curve of the TaqMan real-time PCR method established by using the constructed recombinant plasmid as a standard has a good linear relationship(R2=0.998),and the minimum detection limit of ASFV nucleic acid is 1.3 copies,and there is no cross-reaction with various pathogens such as pseudorabies virus,porcine parvovirus,and porcine circovirus type 2.The real-time quantitative PCR assay for K196R gene established in this study provides a novel,sensitive and specific early detection method for African swine fever.

关 键 词：非洲猪瘟病毒 实时荧光定量PCR K196R基因 检测 

分 类 号：S855.5[农业科学—临床兽医学]