人端粒酶催化亚基(hTERT)实时荧光定量PCR标准品的构建

Construction of a standard plasmid for detecting hTERT with real-time PCR

目的:构建用于检测人端粒酶催化亚基(hTERT)实时荧光定量PCR标准品。方法:通过逆转录PCR扩增得到目的片段后连接至pMD18-T载体,转化DH5α感受态细胞。分别经PCR和测序验证重组质粒的正确性。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梯度稀释制得质粒标准品。10倍梯度稀释质粒标准品后进行实时荧光定量PCR分析。结果:人端粒酶催化亚基(hTERT)基因片段成功克隆至pMD18-T载体之中,重组质粒序列完全正确。实时荧光定量PCR分析10倍梯度稀释的质粒标准品所得标准曲线良好。结论:成功构建了人端粒酶催化亚基(hTERT)实时荧光定量PCR标准品。

Objective：To construct a recombinant plasmid as the standards for hTERT detection by real-time PCR.Methods：Total RNA was extracted from Hela.A fragment of hTERT was amplified by reverse transcription PCR.The PCR production was ligated with pMD18-T vector and transformed to DH5α.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 PCR with ten-fold serially dilution of recombinant plasmid.Results：A fragment of hTERT was successfully cloned into pMD18-T.Dictated by real-time PCR,standard curve could be received from the concentration of 108 to 102 copys/μl.Conclusion：The standard plasmid for detecting hTERT with real-time PCR has been constructed successfully.