人线粒体DNA荧光定量PCR检测方法的建立.docVIP

人线粒体DNA荧光定量PCR检测方法的建立作者：王学波，李建远????作者单位：青岛大学医学院附属烟台毓璜顶医院中心实验室，山东 烟台 【摘要】? 建立SYBR Green I实时荧光PCR定量检测人线粒体DNA的方法。选取人线粒体DNA高度保守基因片段，将该基因片段与pCF-T载体连接后，转化入E.coli DH5α，提取重组质粒PCR及测序鉴定后，作为阳性模板建立SYBR-Green I荧光定量PCR标准曲线和熔解曲线。结果表明：构建的标准曲线线性关系良好(反应体系中含101～108拷贝时,扩增反应CT值与拷贝数的对数成线性关系)，相关系数为0.997。批内和批间重复性测定的变异系数分别为1.23%～3.29%以及3.10%～5.21%。我们成功建立了实时荧光定量PCR检测人线粒体DNA的方法，该方法可作为进一步研究线粒体DNA的方法，在相关疾病诊断和监测中具有一定的应用前景。 【关键词】? SYBR Green I；荧光定量PCR；线粒体DNA；聚合酶链反应；人 ?Establishment of the Method for Detecting Human Mitochondrial ??? DNA with Fluorescence Quantitative Ploymerase ChainWANG Xuebo，LI Jianyuan ??? （The Affiliated Yantai Yuhuangding Hospital of Qingdao Medical University，Yantai 264000，China） ??? Abstract：To estiblish a SYBR-Green I fluorescent quantitative PCR method to detect human mitochondrial DNA.Human mitochondrial DNA was analyzed and special primers were designed and synthesized according to the conserved gene sequence. The specific fragment was cloned into pCF-T vector which was transformed into E.coli DH5α. The positive recombinant plasmid identified by sequencing was used as quantitative template to generate standard curve and melt curve. Results showed that the standard curve succesfully showed a linear relationship between cycle threshold(Ct)and template concentration ranging from 101 to 108 copies,and the correlation coefficient was 0.997.The coefficient of variation values for both intra-experimental and inter-experimental reproducibility ranged from 1.23% to 3.29 % and 3.10% to 5.21%, respectively.It means that the method for detecting human mitochondrial DNA with fluorescence quantitative polymerase chain reaction is developed successfully.It shows good repeatability and sensitivity in detection of human mitochondrial DNA. This method can facilitate the potential applications in the fields of laboratory diagnosis and detection. ??? Key words：SYBR Green I; Fluorescent qunantitative PCR; Mitochondrial DNA；Polymerase chain reaction；Human ??? 1? 引? 言 ??? 线粒体作为真核细胞中一种重要而独特