Abstract：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.
