目的  评价COLD-PCR-HRM 检测结直肠癌患者外周血KRAS 基因突变的临床应用价值。方法  将已知突变型 KRAS 组织DNA 与野生型DNA 做系列稀释，突变DNA 所占比例分别为50%、25%、10%、5%、3%、2% 和 1%。分别应 用常规PCR-HRM 法和COLD-PCR-HRM 法对不同比例KRAS 突变DNA 样本进行检测，验证两种检测方法的灵敏度；同 时应用 COLD-PCR-HRM 法对 62 例外周血和肿瘤组织配对样本进行一致性验证。结果  PCR-HRM 法最低检测浓度为 3%； COLD-PCR-HRM 法最低检测浓度为1%，两种方法的检测灵敏度均明显高于直接测序法（P ＜ 0.05）。应用COLD-PCRHRM 法检测两种样本中KRAS 基因突变状态，其中血清样本中检测出13 例突变（突变率21.0%，13/62），组织中检测出 12 例突变（突变率 19.4%，12/62），两种标本同时存在突变的有 9 例，以组织中检测的 KRAS 状态为准，两者突变一致性 为 75.0%（9/12）。KRAS 基因突变在组织与外周血中存在一致性（κ=0.649；P ＜ 0.001）。结论  COLD-PCR 结合 HRM 明 显提高了 KRAS 突变检测灵敏度，为其应用于低丰度突变样本检测提供了有利条件。

Objective To evaluate the value of COLD-PCR-HRM in detecting KRAS gene mutations in peripheral blood of patients with colorectal carcinoma. Methods DNA from known mutation type was serially diluted into wild-type DNA to the following percentages: 50%, 25%, 10%, 5%, 3%, 2% and 1%. Tasting KRAS gene mutations in different proportions of DNA by conventional PCR-HRM and COLD-PCR-HRM to validate the sensitivity of two testing methods. At the same time, 62 cases of peripheral blood and tumor tissue matching samples were tested by COLD-PCR-HRM for consistency verification. Results The method of PCR-HRM minimum detectable concentration was 3%, and COLD-PCR-HRM was 1%, both methods were significantly higher than that of direct sequencing (P<0.05). KRAS mutations detection in two kinds of sample by COLD-PCR-HRM: 13 cases mutations in serum samples (mutation rate 21.0%, 13/62), 12 cases mutations in tumor tissue (mutation rate 19.4%, 12/62), 9 cases mutations in both two kinds of samples. Mutations in tumor tissue shall prevail, two mutations uniformity was 75.0% (9/12). There were consistency in KRAS mutations in tissue and peripheral blood (κ=0.649; P<0.001). Conclusions COLD-PCR-HRM significantly improved detection sensitivity of KRAS mutations, which provided favorable conditions for its application in low abundance mutation samples.