ABO血型基因分型中靶向捕获二代测序技术的建立  

Establishment of targeted-capture-based next generation sequencing in ABO genotype

作　　者：梁爽 吴凡[1] 苏宇清[1] 梁延连[1] 洪文旭[1] 徐筠娉[1] LIANG Shuang;WU Fan;SU Yuqing;LIANG Yanlian;HONG Wenxu;XU Yunping(Institute of Transfusion Medicine,Shenzhen Blood Center,Shenzhen 518035,China)

机构地区：[1]深圳市血液中心输血医学研究所,广东深圳518035

出　　处：《中国输血杂志》2020年第9期881-885,共5页Chinese Journal of Blood Transfusion

基　　金：中国输血协会威高基金(CSBT-WG-2018-09);深圳市卫生系统科研项目公共卫生防控策略研究(SZGW2018008);深圳市医疗卫生三名工程(SZSM2O1811092);深圳市输血医学重点学科(SZXK070)。

摘　　要：目的建立以多重PCR捕获为基础的ABO血型基因二代测序(next-generation sequencing,NGS)分型技术,结合血清学鉴定技术,对ABO血型准确定型。方法开发用于ABO血型的多重PCR捕获的NGS技术,靶向捕获ABO基因全序列建立DNA文库,利用Illumina NGS平台对20例深圳市血液中心健康无偿献血者血液标本做ABO基因测序分型;同时用血型血清学方法、PCR-SSP基因分型方法、以及第6、7外显子Sanger测序对同一批标本做ABO血型鉴定,以验证这种NGS技术做ABO血型鉴定的准确性。结果 1)利用新建立的靶向捕获二代测序技术对20例健康无偿献血者血液标本ABO基因的测序分析:平均测序深度为4 667±893,特异性与均一性分别为92.59%和92.47%,测序深度>20覆盖度为98.25%,均得到了唯一的分型结果。2)新建立的靶向捕获二代测序技术对于20例标本做的ABO全基因测序分型所得结果与血清学鉴定方法和基因分型技术的结果相符。结论成功建立了ABO基因多重PCR靶向捕获NGS测序技术,可以满足ABO血型的鉴定要求。Objective To establish a targeted-capture-based next generation sequencing(NGS) approach, in combination of routine serology test, aimed at acquiring the accurate ABO blood group typing results.Methods We developed the multiplex PCR technique with customized capture probes targeting the entire ABO gene of 20 healthy voluntary blood donors in Shenzhen Blood Center for sequencing library construction, and then sequenced on Illumina NGS system. Routine serology test, PCR-SSP genotyping and Sanger sequencing of exon 6 and 7 were conducted in parallel, for verifying the accuracy of NGS typing results. Results 1) The new-established targeted-capture-based NGS typing results revealed that the average sequencing depth was 4 667±893 fold, and the rates of specificity and homogeneity were 92.59% and 92.47%, respectively. Besides, 98.25% of the sequence of ABO gene was covered above 20-fold, and all of the samples detected have been draw the unique typing result. 2) The genotyping results of ABO using the newly established NGS method were in full accordance with those from traditional serology and another two genotyping tests. Conclusion In this study, the reliable ABO genotyping has been established successfully with the targeted-capture-based NGS approach.

关 键 词：ABO血型鉴定 基因分型 二代测序 多重PCR捕获 全基因测序 血清学 PCR-SSP Sanger测序 

分 类 号：R457.11[医药卫生—治疗学]