关键词: 电化学发光PCR, 膀胱癌, H-ras癌基因, 点突变

Abstract: 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.

Key words: Electrochemiluminescence polymerase chain reaction(ECL-PCR), Bladder cancer, H-ras oncogene, Point mutation