TaqMan实时荧光定量PCR检测外周血中survivin  

Detection of surivinin Peripheral Blood Using a TaqMan Real-Time Fluorescent Quantitative PCR

作　　者：齐自慧 杨梦歌 王美玉[1] 王业富[1] 

机构地区：[1]武汉大学生命科学学院/病毒学国家重点实验室,湖北武汉430072

出　　处：《武汉大学学报（理学版）》2016年第4期350-354,共5页Journal of Wuhan University:Natural Science Edition

基　　金：湖北省自然科学基金(2005ABA098;204/219100175)资助项目

摘　　要：通过TaqMan实时荧光定量PCR（聚合酶链式反应）,建立一种快速检测凋亡抑制蛋白survivin基因的特异检测方法.选择survivin基因的保守序列设计引物和对应的探针,经PCR扩增95bp序列,经pES1002质粒克隆后转入大肠埃希氏菌E.coli DH5α,提取重组质粒,鉴定后作为模板制作TaqMan实时荧光定量PCR标准曲线,然后进行方法的重复性和灵敏度实验.结果表明,该检测方法能够检测外周血survivin基因,线性范围为1×10^7～1×10^4copies/μL,相关系数为0.998,灵敏度为1×10^2copies/μL.This work aims to develop a fast,sensitive and specific method for the detection of survivinin peripheral blood using the TaqMan real-time fluorescent quantitative PCR（polymerase chain reaction）method.Based on the conserved sequence of survivin,primers and probe were designed.And then,a PCR assay was established.The 95 bp fragment was amplified and cloned into pES1002 vector which was subsequently transformed into E.coli DH5α.After being extracted and identificated,recombinant plasmid was used as quantitative template to generate standard curve.Reproducibility and specificity of the assay was determined as well.The results demonstrated that the linear range of the standard curve was 1×10^7-1×10^4 copies/μL,and the correlation coefficient was 0.998,the sensitivity was 1×10^2copies/μL.

关 键 词：TaqMan实时荧光定量PCR SURVIVIN 肿瘤 外周血 

分 类 号：R730.43[医药卫生—肿瘤]