靶向山羊痘病毒GPCR基因SYBR Green荧光定量PCR检测方法的建立  

The Establishment of a SYBR Green Fluorescence Quantitative PCR Detection Method by Targeting the G-protein Coupled Chemokine Receptor Gene of Goat Poxvirus

作　　者：张红强 任善会 姚威 龚真莉[2] 杨雪[1] 马春玲 尤婷 张玉哲 柳民意 钱文洁 李柳杨 余志鹏 孙跃峰[2] 陈豪泰[2] 樊江峰[1] ZHANG Hongqiang;REN Shanhui;YAO Wei;GONG Zhenli;YANG Xue;MA Chunling;YOU Ting;ZHANG Yuzhe;LIU Minyi;QIAN Wenjie;LI Liuyang;YU Zhipeng;SUN Yuefeng;CHEN Haotai;FAN Jiangfeng(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;Key Laboratory of Veterinary Etiological Biology,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Scicences,Lanzhou 730046,China;Wanzhou Center for Animal Husbandry Industry Development,Chongqing 404100,China)

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]中国农业科学院兰州兽医研究所,兰州730046 [3]重庆市万州区畜牧产业发展中心,重庆404100

出　　处：《畜牧兽医学报》2024年第10期4779-4784,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(32302850);甘肃省科技计划资助(22JR5RA035);甘肃省科技重大专项(22ZD6NA001);中国农业科学院兰州兽医研究所基本科研业务费(1610312021008)。

摘　　要：基于山羊痘病毒(goat pox virus,GTPV)的基因组序列,建立靶向G蛋白偶联趋化因子受体(G-protein-coupled chemokine receptor,GPCR)基因的荧光定量PCR检测方法。基于GTPV全基因序列设计6对qPCR引物,并进行特异性和灵敏性的筛选和检测,最终筛选得到1对高特异性和灵敏性的荧光定量PCR引物;根据GTPV/AV41疫苗株GPCR基因序列,设计普通PCR引物,扩增GPCR基因,构建真核表达载体,建立荧光定量PCR标准曲线。结果表明:筛选得到1对靶向于GPCR基因的特异性qPCR引物;构建pCAGGS-GPCR真核表达质粒,建立标准曲线y=-3.5289x+49.07,线性相关系数R^(2)=0.9972,扩增效率为92.7%;验证性试验结果显示,该引物最低检测限为2.0拷贝·μL^(-1),各批次内与批次间重复性结果的变异系数均小于2%,表明其具有特异性强、重复性好和灵敏度高的优点。建立了靶向山羊痘病毒GPCR基因的qPCR检测方法,为防控山羊痘提供了高效的检测手段。Based on the genomic sequence of the goat pox virus(GTPV),we have established a fluorescence quantitative PCR method targeting the G-protein-coupled chemokine receptor(GPCR)gene.Six pairs of specific qPCR primers were designed based on the whole genomic se quence of GTPV.The specificity and sensitivity of these six qPCR primers were screened and de-tected.A pair of qPCR primers with high specificity and sensitivity was finally screened and ob-tained.According to the GPCR gene sequence of the GTPV AV41 vaccine strain,we designed an ordinary PCR primer pair to amplify the GPCR gene to construct the eukaryotic expression vec-tor,which was used to establish the standard curve.Results showed that A pair of specific qPCR primers targeting the GPCR gene was obtained.The eukaryotic expression plasmid named pCAGGS-GPCR was constructed.The standard curve of y=-3.5289x+49.07 was established,whose linear correlation coefficient is R^(2)=0.9972 and amplification efficiency is 92.7%.The re-sults of the replication experiment suggested that the lowest detective limitation of this primer was 2.0 copies·μL^(-1),and the coefficient variation of the reproducibility results within and be-tween batches was 2%,indicating the advantages of reasonable specificity,repeatability,and sensitivity.We have successfully established a specific fluorescence quantitative PCR method tar-geting the GPCR gene of GTPV,which provides adequate detecting support for the veterinary clinical diagnosis and the prevention and control of goat poxvirus.

关 键 词：山羊痘病毒 GPCR 荧光定量PCR 

分 类 号：S852.654[农业科学—基础兽医学]