人线粒体DNA荧光定量PCR检测方法的建立

生物医学工程研究Journal of Bi om edical Engi neering R es earch

2008,27(4):298～301△通信作者　x @63人线粒体DNA 荧光定量PCR 检测方法的建立

王学波△,李建远

(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　264000)

摘要:建立SY BR G reen I 实时荧光PCR 定量检测人线粒体DN A 的方法。选取人线粒体D NA 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.c oli DH5α,提取重组质粒P CR 及测序鉴定后,作为阳性模板建立SY BR -G reen 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D NA 的方法,该方法可作为进一步研究线粒体DN A 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:SY BR G reen I ;荧光定量PCR ;线粒体DN A ;聚合酶链反应;人

中图分类号:R318 文献标识码:A 文章编号:167226278(2008)0420298204

Establishment of the Method for Detecting H um an Mitochondr ial

D N A w ith Fluor escence Q uantitative Ploymerase Cha in

WANG X uebo ,L I Jianyuan

(The A ffiliated Yantai Y uhuangd ing Ho sp ital o f Qingdao Medical Univ ersity ,Y antai 264000,Ch ina )

A bstr act :T o es tiblish a SY BR -G reen I flu orescent quan titative PCR meth od t o detect human mitoch ondrial DN A.Hu man m it och ondrial D NA w as analyzed and special primers w ere designed and syn thesized accordin g t o the conserved gene sequence.T he s peci fic frag ment was

cloned into pCF -T v ector which was trans formed in to E.coli DH5

α.T he pos itive recomb inant plas mid id entified b y sequen cin g was used as quantitative template to g enerate standard curve and melt curve.Results sh ow ed that the standard curv e succes f u lly sh ow ed a linear relationsh ip betw een cycle thresh old (C t )and tem plate concen tration ran gin g from 101to 108

co pies ,and the correlation coefficien t was 0.997.T he coefficient o f variation values for b oth intra -ex perimental and in ter -experimental reproducibility ranged from 1.23%to 3.29%and 3.10%t o

5.21%,res pectiv ely.It means that the meth od for detecting human mitochondrial D N A with flu orescence quantitative polymerase chain reacti on is devel oped success f u lly.It sh ows g ood repeatability and sensitivity in detection o f human m it och ondrial DN A.Th is method can facilitate the potential applications in the fields of laboratory diagn os is and detection.

K ey w or ds :SY BR G reen I ;Flu orescen t qunantitativ e PCR ;Mitochondrial D NA;Polymerase chain reaction ;Human 1　引　言

线粒体作为真核细胞中一种重要而独特的细胞

器,其性状和数量的改变与人的疾病密切相关。研

究线粒体与疾病的关系,既有理论意义,在医学上也

有潜在的应用前景。称为细胞动力工厂的线粒体,

因其生命周期有限,具有高的更新率,形态、大小、数量和分布常因细胞种类不同而异。本研究旨在建立人线粒体D N A 荧光定量PCR 方法,为进一步研究线粒体D NA 与人的疾病的关系奠定基础。1　材料与方法1.1　实验材料

人EDTA 抗凝血2ml ,菌种为本室保存的大肠

E mail :ueb ow ang https://www.sodocs.net/doc/4e3222081.html,