COLD-PCR-HRM检测结直肠癌患者外周血KRAS基因突变  

Detecting KRAS gene mutations in peripheral blood of colorectal carcinoma with COLD-PCR-HRM

作　　者：李艳丽[1] 赵冬梅[1] 徐育红[1] 张雁[1] LI Yan-li;ZHAO Dong-mei;XU Yu-hong;ZHANG Yan(Department of Clinical Lab, Agricultural Reclamation Bureau General Hospital, Harbin 150088, China)

机构地区：[1]黑龙江省农垦总局总医院检验科,哈尔滨150088

出　　处：《肿瘤代谢与营养电子杂志》2017年第1期51-54,共4页Electronic Journal of Metabolism and Nutrition of Cancer

摘　　要：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%and1%.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,62cases 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 was3%,and COLD-PCR-HRM was1%,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:13cases mutations in serum samples(mutation rate21.0%,13/62),12cases mutations in tumor tissue(mutation rate19.4%,12/62),9cases mutations in both two kinds of samples.Mutations in tumor tissue shall prevail,two mutations uniformity was75.0%(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

关 键 词：KRAS基因突变 COLD-PCR-HRM 结直肠癌 外周血 

分 类 号：R735.34[医药卫生—肿瘤] R730.43[医药卫生—临床医学]