BRCA基因突变检测的标准化研究  

Standardization study on the detection of BRCA gene mutation

作　　者：曲守方[1] 黄传峰[2] 张文新 孙楠[1] 于婷[1] 黄杰[1] QU Shou-fang;HUANG Chuan-feng;ZHANG Wen-xin;SUN Nan;YU Ting;HUANG Jie(Division of Diagnostic for Non-infectious Disease,National Institutes for Food and Drug Control,Beijing 100050,China;Division of Physical and Chemical Testing,National Institutes for Food and Drug Control,Beijing 100050,China)

机构地区：[1]中国食品药品检定研究院非传染病诊断试剂室,北京100050 [2]中国食品药品检定研究院理化检测室,北京100050

出　　处：《中国医药生物技术》2021年第5期439-443,共5页Chinese Medicinal Biotechnology

摘　　要：目的使用BRCA基因突变国家参考品,评价BRCA1/2基因突变检测试剂盒(联合探针锚定聚合测序法)的性能。方法取国家参考品的基因组DNA,进行打断和末端修复,获得片段化的DNA。使用连接酶在DNA两端加上接头。经pre-PCR扩增获得杂交前文库(pre-PCR文库),再用探针特异性捕获含目标区域的DNA片段,经post-PCR扩增获得杂交后文库(post-PCR文库)。使用通用测序试剂盒进行测序,得到原始下机数据。通过数据分析,得到国家参考品的BRCA基因变异信息,从而对试剂盒的准确性、特异性和重复性进行评价。结果试剂盒能够准确检出试剂盒检测范围内的BRCA基因突变位点,并且按照BRCA基因解读规则能够进行正确解读;没有检出其他非标示的致病性或疑似致病性突变位点。使用同一批次试剂盒重复检测国家参考品3次,每次结果均能满足准确性和特异性的要求。结论BRCA基因突变国家参考品可以用于评价BRCA1/2基因突变检测试剂盒(联合探针锚定聚合测序法)的性能,实现对BRCA基因突变检测的标准化。Objective To confirm the national reference material for BRCA gene mutation suitable for evaluating the performance of BRCA gene mutation detection kit(combinatorial probe-anchor synthesis sequencing method).Methods The sequencing library were obtained by genomic DNA fragmentation,end reparation,ligation,pre-PCR amplification,probe capture,and post-PCR amplification according to the manufacture instruction of BRCA gene mutation detection kit(combinatorial probe-anchor synthesis sequencing method)and then sequenced by the universal sequencing reaction kit on BGISEQ-500 sequencing platform.Original sequencing data was analyzed by the BRCA gene analysis software.The accuracy,specificity and reproducibility of the kit were determined based on standard variation information of the national reference material.Results The kit could be used to accurately detect the BRCA gene mutation within the target range,and accurately report the BRCA gene mutation according to the BRCA gene interpretation rules.No other non-labeled pathogenic or likely pathogenic mutation sites were included.The replication results,detection of the national reference for 3 times by the same batch of kit,indicated that the accuracy and specificity of the kit met the requirements.Conclusion The national reference material can be used to evaluate the performance of BRCA gene mutation detection kit,and realize the standardization of BRCA gene mutation detection.

关 键 词：抑癌基因 胚系突变 生物标志物 靶向药物治疗 热点突变 下一代测序 

分 类 号：R737.9[医药卫生—肿瘤] R737.31[医药卫生—临床医学]