血浆循环肿瘤DNA基因突变检测国家参考品的应用评价

Evaluation of the Application of National Reference Materials for Detection of Plasma Circulating Tumor DNA Gene Mutations

目的评价血浆循环肿瘤DNA(ctDNA)KRAS/NRAS/EGFR/BRAF/MET基因突变检测国家参考品。方法对87支国家参考品进行血浆游离DNA提取,文库构建,对文库进行高通量测序,测序后的序列比对到参考基因序列上,通过生物信息软件分析,识别携带基因突变的肿瘤DNA序列,获得相关基因突变信息。结果试剂盒检测范围内的国家阳性参考品均检测出相应突变位点,国家阴性参考品和试剂盒检测范围外的阳性参考品均为野生型,可检测50 ng DNA中突变频率为0.3%~1%的检测限参考品。结论该技术方法的试剂盒检测结果符合国家参考品的要求。本国家参考品为ctDNA检测的标准化提供保障。

Objective To evaluate the national reference material of KRAS/NRAS/EGFR/BRAF/MET gene mutation detection of circulating tumor DNA(ctDNA).Methods After the library construction and specific amplification of national reference material,the amplified products were sequenced with high flux.The sequenced sequences were compared with the reference gene sequences.The original plasma DNA template was analyzed and reduced by bioinformatics software.The tumor DNA sequences carrying gene mutations were accurately identified and the relevant gene mutation information was obtained.Results The corresponding mutation sites were detected in the positive reference materials within the detection range.The negative reference material and the positive reference outside the detection range were wild type.The detection limit reference material with mutation frequency of 0.3%~1% in 50 ng DNA can be detected.Conclusion The test results of this technology meet the requirements of national standard.The national standard provides guarantee for the standardization of ctDNA detection.