小鼠脑脊髓炎病毒非编码蛋白片段实时荧光定量PCR标准品的构建

摘要

目的 构建小鼠脑脊髓炎病毒(TMEV)非编码蛋白(UTR)片段标准品,用于实时荧光定量 PCR检测小鼠脑脊髓炎病毒.方法 RT-PCR 扩增TMEV UTR片段上80~1094 nt之间长度为1 014 bp的片段,将目的 片段连接至pMD18-T载体,转化至DH5a感受态细胞.分别经 PCR鉴定和序列测定验证重组质粒.分光光度计测量重组质粒的吸光值,换算成拷贝数浓度后作10倍梯度稀释制得质粒标准品.然后进行实时荧光定量PCR分析,绘制标准曲线.结果 TMEV UTR片段成功克隆至pMD18-T载体中,测序结果 表明重组质粒中插入的UTR序列正确,实时荧光定量PCR分析10倍梯度系列稀释的质粒标准品所得到的标准曲线良好,统计学分析显示Ct值与标准品浓度的对数存在良好线性关系,回归系数在0.99以上.结论 成功构建了TMEV UTR 片段实时荧光定量PCR标准品,为今后TMEV实时荧光定量PCR检测打下了基础.%Objective To construct a recombinant plasmid as the standard for Theiler's murine encephalomyelitis virus (TMEV)detection by real-time quantitative PCR. Methods A specific TMEV untranslated region (UTR) fragment in size of 1014 bp was amplified by RT-PCR, The PCR product was ligated with pMD18-T vector and transformed into E.coli DH5oc. Recombinant plasmid was identified by PCR and sequencing. The concentration of recombinant plasmid was analyzed through its absorption at 260 nm. Standard curve was constructed by real- time quantitative PCR with ten-fold serial dilutions of recombinant plasmid. Results The fragment of UTR was successfully cloned into pMD18-T vector. The quantitative standard curve of recombinant standard plasmids was obtained, statistics analysis indicated that there was a good linear correlation between the Ct value and the concentration gradient of standard plasmids. The regression value was over 0.99. Conclusion The standard plasmid for detecting TMEV with real-time PCR has been constructed successfully.