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目的:KRAS基因在结直肠癌患者突变率为30%～50%,并且KRAS基因状态与抗EGFR抗体疗效的密切相关。常用的直接测序法其检测灵敏度低、容易出现假阴性结果。采用高分辨率溶解曲线分析法检测结直肠癌中KRAS基因突变,探索其应用于临床检测的可行性。方法:本文收集20例结直肠癌患者石蜡包埋组织标本,应用高分辨率熔解曲线方法检测KRAS基因突变(2,3号外显子)。将KRAS基因突变型DNA(p.G12D)和野生型DNA梯度混合稀释,突变型DNA浓度分别为40%、20%、10%、2%,进行高分辨率熔解曲线方法进行检测,并将扩增产物进行直接测序,比较两种方法的灵敏度。结果:在20例结直肠癌患者中检出5例2号外显子突变,p.G12D 2例,p.G12V 2例,p.G13D 1例,3号外显子未检出突变。HRM方法检测基因突变的检出限可达2%,直接测序法检测基因突变的检出限为20%。结论:HRM法比直接测序法灵敏度高,能精确检测出结直肠癌石蜡标本中低浓度的KRAS突变,有望应用于临床基因突变检测。
Objective: KRAS gene mutation rate in patients with colorectal cancer is 30% to 50%, and KRAS gene status and anti-EGFR antibody efficacy is closely related. Commonly used direct sequencing method of its detection sensitivity is low, prone to false negative results. KRAS gene mutation in colorectal cancer was detected by high resolution lysing curve analysis and the feasibility of its application in clinical detection was explored. Methods: Twenty paraffin-embedded tissue specimens from patients with colorectal cancer were collected and KRAS gene mutations (Exons 2 and 3) were detected by high resolution melting curve method. The KRAS mutated DNA (p.G12D) and wild-type DNA were mixed and diluted. The concentration of mutant DNA was 40%, 20%, 10% and 2%, respectively. The high resolution melting curve method was used for detection. The products were directly sequenced, comparing the sensitivity of the two methods. RESULTS: Five cases of exon 2 mutations were detected in 20 patients with colorectal cancer. There were 2 cases of p.G12D, 2 cases of p.G12V, 1 case of p.G13D, and no mutation of exon 3. The limit of detection of gene mutation detected by HRM method is up to 2%, and the detection limit of gene mutation detected by direct sequencing is 20%. Conclusion: The HRM method is more sensitive than direct sequencing and can accurately detect low concentration of KRAS mutation in paraffin-embedded specimens of colorectal cancer. It is expected to be used in the detection of clinical gene mutations.