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-pro-tein-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R2=0.997 2,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.
Abstract
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 R2=0.997 2 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-getting the GPCR gene of GTPV,which provides adequate detecting support for the veterinary clinical diagnosis and the prevention and control of goat poxvirus.
基金项目
国家自然科学基金(32302850)
甘肃省科技计划(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)
维普
万方数据