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

摘要

基于等位位点特异性扩增的原理,设计锁核酸修饰KRAS基因突变特异性扩增引物,结合封阻探针技术,建立检测KRAS基因突变的荧光定量PCR方法.结果发现,锁核酸修饰的引物及探针可显著提高等位位点特异性扩增技术用于复杂样本中的微量基因突变检测的敏感度,该技术检测KRAS基因突变的敏感性可达0.01％～0.1％.进一步用建立的荧光定量PCR方法检测52例结直肠癌患者血浆标本,并用DNA测序法作为对照,同时用健康人血浆标本建立阴性检测结果判读标准,以初步评价该方法应用于循环DNA中KRAS基因突变检测的可行性.结果发现结直肠癌患者KRAS基因突变主要是G12C、G12A和G12R,而且qPCR法的阳性检出率为46.15％,高于DNA测序法(13.46％),阴性结果与DNA测序法的符合率为100％.此外,结直肠癌患者外周血KRAS基因的突变检出率与文献报道组织标本中的突变检出率及常见突变类型基本相符.上述结果说明该方法检测循环肿瘤DNA (circulating tumor DNA,ctDNA)具有较高的可靠性,可以用于肿瘤患者循环血液中KRAS基因突变的检测.%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.