电化学发光PCR定量检测H-ras癌基因点突变

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100fmol;线性范围为0.1-500pmol.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10μL样品,20min的孵育时间和30s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.

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

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.