生命科学研究2017，Vol.21Issue(3)：189-194,6.DOI:10.16605/j.cnki.1007-7847.2017.03.001
基于锁核酸及等位位点特异性扩增技术的KRAS基因突变检测荧光定量PCR方法研究
KRAS Mutation Detection Method Based on LNA and Allele Specific Amplification
摘要
Abstract
Locked nucleic acid (LNA) modified KRAS mutation specific PCR primers were designed based on the principle of allele specific amplification,and combined with blocking probe technique,the fluorescencebased quantitative PCR method was established to detect mutations in the KRAS gene.The results showed that LNA modified primers and probes could significantly improve the detection sensitivity of trace gene mutations of complex samples by the allele specific amplification technique,and that the sensitivity of this technology to detect KRAS gene mutation was 0.01％～0.1％.Then,the plasma samples of 52 patients with colorectal cancer were detected by the established quantitative fluorescent PCR,and the DNA sequencing was used as the control.Meanwhile,the standard for interpretating the negative results was established with healthy human plasma samples.The results showed that the major mutations of KRAS gene in colorectal cancer patients were G12C,G12A and G12R,and that the positive rate of qPCR was 46.15％,which was higher than that of DNA sequencing (13.46％).While the coincidence rate of negative results between qPCR and DNA sequencing was 100％.Furthermore,the detection rate of KRAS gene mutations in peripheral blood of colorectal cancer patients was basically consistent with the mutation detection rate and the common mutation types in tissue samples reported in the literature.These indicate the method has a high reliability to detect circulating tumor DNA and can be used in the detection of circulating KRA S mutations in tumor paticnts.关键词
结直肠癌/循环肿瘤DNA(ctDNA)/KRAS基因突变检测/荧光定量PCR方法(qPCR)/等位基因扩增/锁核酸(LNA)/分子靶向治疗Key words
colorectal cancer/circulating tumor DNA (ctDNA)/detection of KRAS gene mutation/fluorescence-based quantitative PCR method (qPCR)/allele specific amplification/locked nucleic acid (LNA)/molecular targeted therapy分类
生物科学引用本文复制引用
刘明华,景奉香,李瑶,吴海,张冀申,张亚龙,孙文洁..基于锁核酸及等位位点特异性扩增技术的KRAS基因突变检测荧光定量PCR方法研究[J].生命科学研究,2017,21(3):189-194,6.基金项目
国家自然科学基金面上项目(61271162) （61271162）
