荧光定量PCR检测TRECs的标准品质粒和标准曲线的构建  

Construction of recombinant plasmid and standard curve for detection of TRECS by real-time quantitative PCR

作　　者：李红兵[1] 陈明[1] 朱学文[1] 汪莉萍[1] 葛国洪[1] 李秀华[1] 

机构地区：[1]徐州医学院附属医院感染病科,陕西渭南市中心医院221002

出　　处：《江苏医药》2008年第2期173-175,共3页Jiangsu Medical Journal

基　　金：江苏省科委社会发展计划基金资助项目(BS2004014)

摘　　要：目的构建荧光定量PCR检测T细胞受体重排切除环（TRECs）的标准品质粒和标准曲线。方法对TCRδ基因进行序列分析，设计一对引物和探针。提取正常人外周血单个核细胞中的DNA，经普通PCR扩增，产物纯化后与pUCM-T载体连接并转化入大肠杆菌DH5α，筛选得到重组成功的质粒。结果重组质粒测序后显示目的片段序列正确，表明TRECs基因片段成功克隆。以10^3～10^7copies／ml不同稀释水平的标准品进行荧光定量PCR扩增后，统计学分析显示标准品浓度的对数与Ct值之间存在良好的线性关系（r=-0．998，P＜0．01）。结论所构建的TRECs标准品特异性和线性关系较好。Objective To construct recombinant plasmid and standard curve for the detection of TCR rearrangement excision circles （TRECs）by real-time quantitative PCR assay. Methods The primer expression software was used to obtain a couple of primer and fluorescent probe after analysis of TCRδ gene sequence. DNA was extracted from PBMCs of normal individuals and then amplified by PCR. The PCR product was connected with pUCM-T vector and then transferred into Ecoil DH5α. The standard recombinant plasmid was gained from the positive plasmid. Results The sequence of target segment in recombinant plasmid was confirmed to be correct by sequence check, which indicated that TRECs segment was cloned successfully. There was a good liner function in statistics between the Ct value and the concentration gradient of standard DNA specimen in 10^3-10^7 standard DNA specimen after real-time quantitative PCR amplification（r=-0. 998）. Conclusion The recombined plasmid and standard curve for real-time quantitative PCR to detect TRECs were well both in linearity and speciality.

关 键 词：聚合酶链反应 T细胞受体重排切除环 标准曲线 质粒 

分 类 号：R392[医药卫生—免疫学]