山羊关节炎脑炎病毒SYBR GreenⅠ荧光定量PCR方法的建立及应用  

Establishment and application of SYBR GreenⅠreal-time PCR for CAEV

作　　者：汪伟[1,3] 杜倩 韩知晓 辛佳亮 闫修魁 梁莹莹 朱远致 曹亮 孙文超[3,4] 胡传活 郑敏[2] WANG Wei;DU Qian;HAN Zhi-xiao;XIN Jia-liang;YAN Xiu-kui;LIANG Ying-ying;ZHU Yuan-zhi;CAO Liang;SUN Wen-chao;HU Chuan-huo;ZHENG Min(Animal Science and Technology,Guangxi University College,Nanning 530001,China;Guangxi Center for Animal Disease Control and Prevention,Nanning 530004,China;Institute of Military Veterinary,The Academy of Military Medical Sciences,Changchun 130122,China;Institute of Virology,Wenzhou University,Wenzhou 325035,China)

机构地区：[1]广西大学动物科学技术学院,广西南宁530001 [2]广西壮族自治区动物疫病预防控制中心,广西南宁530004 [3]军事科学院军事兽医研究所,吉林长春130122 [4]温州大学病毒学研究所,浙江温州325035

出　　处：《中国兽医科学》2020年第3期294-299,共6页Chinese Veterinary Science

基　　金：广西自然科学基金项目(2012GXNSFAA053073);浙江省青年基金项目(LQ19C180001)。

摘　　要：为建立检测山羊关节炎脑炎病毒(caprine arthritis-encephalitis virus,CAEV)的快速诊断方法,本研究根据CAEV甘肃株Gag基因设计1对引物,并以含有CAEV甘肃株全基因组序列的质粒pUC57-CAEV为模板,建立CAEV的SYBR GreenⅠ荧光定量PCR检测方法,并验证其特异性、灵敏性和重复性。结果显示,本研究所建立的SYBRGreenⅠ荧光定量PCR检测方法的Ct值与标准品模板在1.19×109~1.19×10^1 copies/μL范围内呈良好的线性关系,相关系数为0.997,斜率为-3.74,检测下限为1.19×10^1 copies/μL,且对痘苗病毒、山羊痘病毒均无扩增。重复性试验结果显示,批间与批内变异系数均小于1%,重复性好。本研究首次建立了CAEVGag基因的SYBRGreenⅠ荧光定量PCR检测方法,为CAEV的快速检测和病毒感染预防提供了技术手段。In order to establish a rapid diagnostic method for the detection of caprine arthritis-encephalitis virus(CAEV).In this study,a pair of primer based on gag gene of CAEV Gansu strain was designed,and a SYBR GreenⅠPCR assay for CAEV was established and its specificity,sensitivity and repeatability was tested,using the plasmid pUC57-CAEV contained the full genomic sequence of Gansu strain the standard template.The test results showed that the Ct value showed a good linear relationship with the standard in the range of 1.19×109-1.19×10^1 copies/μL and the correlation was 0.997 with a slope of-3.74.The detection limit is 1.19×10^1 copies/μL,and there is no specific amplification of vaccinia virus and GTPV.The repeatability test resulted showed that the inter-assay and the intra-assay coefficient of variation were both less than 1%,indicating a good repeatability.This study established the SYBR GreenⅠreal-time PCR detection method for CAEV Gag gene,which provided a technical means for rapid detection of CAEV and control of viral infection.

关 键 词：山羊关节炎脑炎病毒 SYBR GreenⅠ 荧光定量PCR GAG基因 

分 类 号：S852.659.3[农业科学—基础兽医学]