非洲猪瘟病毒SYBR Green I实时荧光定量PCR检测方法的建立  

Development of a SYBR Green I based real-time quantitative PCR method for the detection of African swine fever virus

作　　者：贾云飞 赵福杰 朱静静[2] 王超群 吴亚楠 魏战勇[2,4] JIA Yunfei;ZHAO Fujie;ZHU Jingjing;WANG Chaoqun;WU Yanan;WEI Zhanyong(School of Economics&Management,Henan Agricultural University,Zhengzhou 450002,China;Engineering College of Animal Husbandry and Veterinary Science,Henan Agricultural University,Zhengzhou 450002,China;Xuchang Customs,Xuchang 461000,China;Henan Animal Food Safety Key Laboratory,Zhengzhou 450002,China)

机构地区：[1]河南农业大学经济与管理学院,河南郑州450002 [2]河南农业大学牧医工程学院,河南郑州450002 [3]许昌海关,河南许昌461000 [4]河南省动物性食品安全重点实验室,河南郑州450002

出　　处：《河南农业大学学报》2020年第1期69-73,80,共6页Journal of Henan Agricultural University

基　　金：中国工程院咨询研究重点项目(2020-XZ-016);许昌市基础与前沿项目(JC2018005)。

摘　　要：为了建立非洲猪瘟病毒(African swine fever virus,ASFV)快速、敏感的检测方法,根据GenBank中登录的中国流行株ASFV-SY18的P 72基因,建立了SYBR Green I实时荧光定量PCR检测方法,并对所建立方法的灵敏性、特异性与重复性进行了验证。结果显示,所建立的非洲猪瘟病毒SYBR Green I实时荧光定量PCR检测方法能有效扩增1.0~1.0×109 copies·μL-1的ASFV标准质粒,建立的标准曲线呈现良好的线性关系;检测灵敏度为1.0 copies·μL-1;对猪流行性腹泻病毒、猪δ冠状病毒、高致病性猪繁殖与呼吸综合征病毒、猪伪狂犬病毒等病原不发生交叉反应,具有很好的特异性;重复性试验结果显示变异系数小于1%,重复性良好。In order to establish a rapid and sensitive method for detection of African swine fever virus(ASFV),a SYBR Green I based Real-time quantitative PCR method was developed according to the P 72 gene sequence of the Chinese epidemic strain ASFV-SY18 registered in GenBank,and the sensitivity,specificity and reproducibility of the method were verified.The results showed that the SYBR Green I real-time PCR method could detect the ASFV ranging from 1.0×100 to 1.0×109 copies·μL-1 with good linear relation,and the sensitivity detected was 1.0 copies·μL-1.With good specificity,the method had the advantages of having no cross-reactions to other pathogens such as porcine epidemic diarrhea virus,porcine coronavirus,highly pathogenic porcine reproductive and respiratory syndrome virus,and porcine pseudorabies virus.The reproducibility tests results showed that the coefficient of variation was less than 1%,indicating that the reproducibility was good.

关 键 词：非洲猪瘟病毒 P 72基因 实时荧光定量PCR 检测 

分 类 号：S852.65[农业科学—基础兽医学]