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.
出处
《高等学校化学学报》
SCIE
EI
CAS
CSCD
北大核心
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
关键词
电化学发光PCR
膀胱癌
H—ras癌基因
点突变
Electrochemiluminescence polymerase chain reaction(ECL-PCR)
Bladder cancer
H-ras oncogene
Point mutation