电化学发光PCR定量检测H-ras癌基因点突变  

Quantitative Detection of H-ras Point Mutation in Bladder Cancer by ECL-PCR Method

作　　者：朱德斌[1] 邢达[1] 沈行燕[1] 刘晋峰[1] 

机构地区：[1]华南师范大学激光生命科学研究所,广州510631

出　　处：《高等学校化学学报》2005年第7期1248-1251,M005,共5页Chemical Journal of Chinese Universities

基　　金：国家重大基础研究前期专项基金(批准号:2002CCC00400);国家自然科学基金(批准号:60378043);广东省自然科学基金团队项目(批准号:015012)资助

摘　　要：提出了一种电化学发光PCR(ECL-PCR)分析方法, 该法可用于定量检测基因点突变. 其检测H-ras癌基因PCR扩增产物的灵敏度可达100 fmol;线性范围为0.1～500 pmol. 用ECL-PCR分析法对膀胱癌组织中H-ras癌基因进行突变检测, 只需要10 μL样品, 20 min的孵育时间和30 s的采集时间, 得出20例膀胱癌样品中有7例存在点突变, 通过标准曲线方程定量计算出突变样品的量. ECL-PCR分析方法在灵敏度、线性范围、分析时间等方面都优于传统的检测方法, 是一种安全、快速、灵敏、定量检测基因点突变的分析方法.The ability to detect point mutation is of great importance in molecular genetics. However, conventional electrophoresis-based methods often require long time with multi-step, using radioactive isotopes or other hazardous materials, and the result is only semi-quantitive. In this study, an electrochemiluminescence polymerase chain reaction(ECL-PCR) method for quantitative detection of H-ras point mutation was devoloped. The results show that the sensitivity of the assay for H-ras amplicon was 100 fmol and the linear range was from 0.1 to 500 pmol. Twenty bladder cancer samples were tested by using the ECL-PCR assay. The positive rate of H-ras point mutation was 35%. Mutant H-ras amplicon was then quantified according to the calibration curve. In the assay, only 10 μL of sample was used for each test and a 20 min incubation period was required in addition to a 30 s acquisition time. It is significantly better in terms of safety, sensitivity, linear range, and assay time, compared with the traditional methods. The results of the study suggest that ECL-PCR is a feasible quantitative method for rapid screening of a large amount of clinical samples.

关 键 词：电化学发光PCR 膀胱癌 H—ras癌基因 点突变 

分 类 号：O657[理学—分析化学]