构建hnRNP B1相对定量的FQ RT-PCR检测方法  

A Method for Detecting hnRNP B1 mRNA by Fluorescence Quantitative Reverse Transcriptase Polymerase Chain Reaction

作　　者：常珍[1] 沈佐君[2] 操乐杰[3] 

机构地区：[1]安徽医科大学附属安徽省立医院检验科,合肥230001 [2]安徽省立医院临检中心 [3]安徽省立医院呼吸科

出　　处：《临床输血与检验》2007年第2期111-114,共4页Journal of Clinical Transfusion and Laboratory Medicine

基　　金：安徽省自然科学基金(No.00044517);安徽省重点科研项目计划(No.01023024)资助

摘　　要：目的建立一种荧光定量逆转录聚合酶链反应(FQ RT-PCR)检测肺癌患者外周血hnRNP B1mRNA表达的相对定量方法。方法以hnRNP B1为待测基因,以β-actin为参照进行检测,建立标准曲线,确定实时RT-PCR的扩增效率;优化反应条件,使扩增效率接近100%,检测待检标本hnRNP B1和β-actin的循环阈值(Ct)。结果该法检测的最低拷贝数为103,线性范围为103～107拷贝,批内和批间变异系数(CV)分别为3.4%和11.3%。结论该方法具有灵敏、特异、数据处理简便可靠等特点,为其他肿瘤患者外周血中肿瘤细胞的检测提供了方法学启示。Objective To develop a fluorescence quantitative reverse transeriptase polymerase chain reaction （FQ RT-PCR） method for detecting hnRNP B1 mRNA in peripheral blood of lung eaeinoma patients. Methods hnRNP B1 was used as target gene, and β-aetin as reference. Stand curves were established to measure the amplification efficiency of hn-RNP B1 and β-aetin. By changing reaction conditions,the amplification efficiency of two gene was nearly 100%. Ct values of hnRNP B1 and β-aetin were measured in samples stimultaneously. Results The method could detect as low as 103 copies and the linear range was 10^3- 10^7 copies,the intra-assay variation was 3.4 % ,the inter-assay variation was 11.8%. Conclusions The method is a sensitive,specific procedure with simple and reliable data analysis, which providing a promising method for the detection of follow-up patients of different kinds of cacinoma.

关 键 词：荧光定量聚合酶链反应 异质性胞核核糖核蛋白B1 肺癌 

分 类 号：Q7[生物学—分子生物学] R734.2[医药卫生—肿瘤]