一步法实时荧光定量PCR检测白血病WT1基因mRNA.pdfVIP

· 290· 南方医科大学学报(J South Med Univ) 2008；28(2) 一 步法实时荧光定量PCR检测白血病WT1基因mRNA 许文娟，徐 兵，李 冰(南方医科大学南方医院血液科，广东 广州 510515) 摘要：目的 构建RT．PCR一步法实时荧光定量PCR检测WTl基因mRNA表达的方法．准确定量检测白血病患者微 小残留病，指导临床判断预后和早期预测复发。方法 从K562细胞提取的RNA用特异性引物扩增WTl基因．pMD l8．T载体克隆法构建荧光定量PCR标准模板，建立RT．PCR一步法实时荧光定量PCR检测WTl基因方法．并对该方 法的灵敏度、重复性和稳定性进行检测。结果 构建的RT．PCR一步法实时荧光定量PCR方法的灵敏度达 104水平：以 拷贝数为 1．0xlM．0xl02 cps／ml的标准品，分别进行RT．PCR一步法实时荧光定量PCR扩增后，标准品的Ct值与该 标准品模板起始浓度的对数存在线性关系，r值达0．998；管间和批间的变异系数均小于8％。结论 所建立RT．PCR一 步法实时荧光定量PCR检测WTl基因方法的灵敏度、重复性和特异性好；RT和PCR反应过程一步完成，更简便快 捷，减少加样和污染机会，更适合临床检测。 关键词：聚合酶链反应定量；白血病；WTl基因 中图分类号：R733 文献标识码：A 文章编号：l673-4254(2008)02．0290．03 One-step real-time fluorescence quantitative PCR for detecting WT1 mRNA expression in leukemia XUWen-juan，XUBing，LIBing Department ofHematology，Nanfang Hospital，Southern Medical University，Guangzhou 510515，China Abstract： Objective To establish a one-step real-time quantitative RT—PCR assay for detecting the expression ofⅥ l mRNA．which allows detection ofthe minimal residue disease and prognostic prediction in leukemic patients．MethodsⅥ l gene fragment was amplified from the RNAs extracted from K562 cells using one—step RT—PCR． The quantitative standard were constructed by pMD 1 8一T vector cloning， and a Taqman—MGB fluorescent probe and a pair of primers were used to establish the one—step real-time fluorescence quantitative RT—PCR assay forⅥ l gene detection．The sensitivity．repeatability , and stability ofthis assay were evaluated and verified． Results The sensitivity ofthis assay reached the l 0r4 leve1． The standard template of 1．0xl-I．0xl0 copies／ml were am plified by the one-step real-time fluorescence quantitative RT—PCR assay，and the Ct value was strongly correlated(r=0．998)to the logarithm ofthe initial template concentration．The repetition