荧光定量聚合酶链反应检测基质金属蛋白酶组织抑制因子-1方法的建立  

A sensitive, specific method of real-time quantitative PCR for detection of tissue inhibitor of metalloproteinase-1 gene

作　　者：丛敏[1] 阎钟钰[1] 王萍[1] 张岩[1] 徐雍[1] 卢炎[1] 王宝恩[1] 贾继东[1] 尤红[1] 

机构地区：[1]首都医科大学附属北京友谊医院肝病中心,100050

出　　处：《中华检验医学杂志》2005年第5期533-537,共5页Chinese Journal of Laboratory Medicine

基　　金：北京市科技新星计划(2004B32);北京市优秀人才培养专项经费资助项目(2004200300104)

摘　　要：目的构建含有金属蛋白酶组织抑制因子-1(TIMP-1)的重组质粒并以其为模板建立荧光定量聚合酶链反应(PCR)技术检测TIMP-1的标准曲线,为荧光定量PCR准确检测TIMP-1奠定基础.方法提取大鼠星状细胞系HSC-T6的mRNA,经RT-PCR扩增TIMP-1基因后与pGMT-Vector 连接,转化到大肠杆菌DH5α,以筛选得到的阳性克隆质粒作为标准品进行梯度稀释,分别用Taqman荧光探针技术和SYBR Green I荧光染料技术进行荧光定量PCR检测,建立标准曲线,并对两者结果进行了对比.同时进行普通PCR检测,与荧光定量PCR方法检测结果进行比较.结果重组质粒经PCR扩增及序列测定,表明pGMT-TIMP-1基因已成功克隆.以不同稀释水平的标准品质粒进行荧光定量PCR扩增, 应用Taqman荧光探针技术建立的标准曲线的线性检测范围为7×104～7×108拷贝,检测灵敏度为7×104拷贝;应用SYBR Green I荧光染料技术的线性检测范围为7×106～7×108拷贝,检测灵敏度为7×106拷贝,且熔解曲线显示出现非特异性扩增;两种技术相对于普通PCR技术均具有定量功能.结论所构建的pGMT-TIMP-1基因荧光定量PCR检测标准品应用Taqman荧光探针技术建立的标准曲线线性关系好, 灵敏度和特异性高,准确可靠,此方法可作为荧光定量PCR检测TIMP-1基因的标准方法.Objective To construct the recombinant plasmid and standard curve for detection of tissue inhibitor of metalloproteinase-1 (TIMP-1) gene by real-time quantitative PCR (QPCR) and establish the real-time QPCR assay for accurate detection of TIMP-1.Methods The TIMP-1 gene was acquired by reverse transcription-PCR after isolating mRNA from rat hepatic stellate cells (HSC). The purified product of PCR was subsequently ligased with pGMT-Vector and transferred into Ecoli DH5α. The standard recombinant plasmid was gained from the positive clone.The gradient diluted recombinant plasmid were used as the template to amplify the TIMP-1 gene by two methods, fluorescence QPCR by “Taqman” approach and by SYBR Green I approach.The standard curves made by the two methods were compared and the results of the two methods were compared with the conventional PCR.Results The amplified products of the recombinant plasmid by PCR and gene sequencing confirmed that the pGMT-TIMP-1 gene was successfully cloned. When using the gradient diluted recombinant plasmid as the template to amplify the TIMP-1 gene by the two real-time QPCR,“Taqman” approach can detect 7×10 4～7×10 8 copies , while SYBR Green I approach only detect 7×10 6～7×10 8 copies. The sensitivities of two methods were 7×10 4 copies and 7×10 6 copies, respectively. The melting curve also showed that unspecific amplification appeared when detected by SYBR Green I approach. Meanwhile, both of the two real-time QPCR have the quantitative function compared with the conventional PCR.Conclusion The recombinant plasmid and standard curve to detect the TIMP-1 gene by the “Taqman” approach was good at sensitivity,specificity,quantification and linear function. It can be a standard method of real-time QPCR for detection of TIMP-1.

关 键 词：基质金属蛋白酶组织抑制因子-1 荧光定量聚合酶链反应检测 Taqman荧光探针技术 荧光定量PCR检测 TIMP-1基因 聚合酶链反应(PCR) 荧光定量PCR方法 RT-PCR扩增 Green 检测灵敏度 标准曲线 HSC-T6 重组质粒 SYBR 荧光染料 

分 类 号：R446.9[医药卫生—诊断学]