HRM法检测结直肠癌组织KRAS基因密码子12和13点突变

High resolution melting analysis for the rapid and sensitive detection of KRAS codon 12 and 13 mutations in colorectal cancer

目的建立HRM法检测大肠癌患者肿瘤组织KRAS（v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog）基因突变的方法。方法采用HRM法对含不同比例KRAS基因突变型质粒的系列混合样本进行检测，以评价其灵敏度。应用HRM法检测60份大肠癌患者新鲜肿瘤组织KRAS基因密码子12和13的突变状况，并与直接测序法的结果进行比较分析。结果HRM法只需在PCR结束后直接运行高分辨熔解，即可获得检测结果。HRM法可检出系列混合样本中突变型质粒比例为10％的突变，其检测灵敏度达10％。HRM法从60份大肠癌患者组织标本中，检出17份KRAS基因密码子12或13突变（28．3％）；直接测序法检出15份（25．0％）突变，2份未检出KRAS基因突变。HRM法检测的敏感度为100％（15／15），特异度为96％（43／45）。结论HRM法在筛选大肠癌标本的KRAS基因突变类型时，具有操作简便、快速、灵敏，单管避免污染等优点，完全符合临床个体化治疗的要求，值得推广。

Objective To establish a HRM assay to screen for KRAS mutations in clinical colorectal cancer patients. Methods The sensitivity of HRM was analyzed by detecting somatic mutations in exon 2, notably codons 12 and 13 of the KRAS gene in the serial plasmid mixture samples which were mixed using the different proportions mutation plasmid and wide type plasmid of KRAS. HRM analysis was performed for KRAS on DNA insolated from a panel of 60 colorectal cancer samples derived from fresh tissues. The results were compared with the direct sequencing data. Results After the PCR amplification, the mutation results could be available by performing HRM analysis in the same tube on a real time PCR machine with HRM capability. HRM detection could identify KRAS mutation in a proportion of 10% of mutation plasmid DNA. All 60 samples identified the KRAS mutation by HRM and sequencing. 17 samples were positive（28.3% ）by HRM for KRAS exon 2 mutations, and 15 samples were confirmed the presence of codon 12 or 13 mutations （25.0%） and the other 2 samples were wild type by sequencing. The 60 samples detected by HRM were given 100% sensitivity with 96% specificity. Conclusions HRM is a sensitive in- tube methodology to screen for mutations in clinical samples. HRM will enable high-throughput screening to gene mutations to allow appropriate therapeutic choices for patients and accelerate research aimed at identifying novel mutations in human cancer.