人类血小板抗原全套基因分型方法的建立与应用

Establishment and application of human platelet antigen genotyping with PCR sequencing-based typing method

目的建立一种基于DNA扩增和测序分型（PCR sequencing—based typing，PCR—SBT）的人类血小板抗原HPA-1～HPA-16w的基因分型方法。方法从免疫多态性数据库中下载HPA-1-HPA-16w的全套HPA核酸序列，以Primer Premier 5．0软件设计引物，特异性扩增包含HPA—1a／1b到HPA-16aw／16bw多态性的核酸片段。通过调整引物序列和PCR条件获得单一的特异性扩增产物，PCR产物纯化后直接进行DNA序列分析并确定HPA基因型。通过对2份标准样本的HPA分型验证PCR-SBT方法的准确性；并利用PCR-SBT法对2008年度国际输血协会（ISBT）第14届国际血小板免疫学工作组提供的16份比对样本（包括6个含有基因突变的干扰样本）进行HPA基因分型。结果设计11对引物扩增和测序16个HPA系统。2份标准样本的HPA基因型分别为HPA：1aa／2aa／3ab／4aa／5ab／6aa．／7aa．／8aa／9aa／10aa／11aa／12aa／13aa／14aa／15aa／16aa和HPA：1aa／2aa／3aa／4aa／5aa／6aa／7aa／8aa／9aa／10aa／11aa／12aa／13aa／14aa／15aa／16aa。16份ISBT考核样本的256个HPA基因型结果清晰，其中HPA-1～HPA-6w、HPA-9w和HPA-15共8个系统的128个基因型结果与ISBT参考结果完全一致。结论建立了HPA-1～HPA-16w的PCR—SBT分型方法，结合高通量的DNA序列分析仪，具有简单、快速和准确的优点，适合于临床HPA全套基因分型，具有良好的应用前景。

Objective To establish a PCR sequencing-based typing （PCR-SBT） method for simultaneous genotyping of human platelet antigen HPA-1 to HPA-16w. Methods All DNA polymorphism sites of HPA-1 to HPA-16w were obtained from the immuno polymorphism database. The specific primers were designed using Primer Premier 5.0 software to amplify nucleotide acid fragments encompassing each HPA polymorphism site. The primer sequence and PCR condition were optimized to obtain specific and single amplification product. The PCR product was purified and then sequenced to determine the HPA genotypes. Two standard DNA samples were detected using the HPA PCR-SBT method to examine the accuracy of this method. Sixteen reference samples （including 6 interference samples with HPA gene mutations） provided by 14th platelet immunology workshop of international society of blood transfusion （ISBT） in 2008 were also tested by this home-brew HPA PCR-SBT method. Results Total eleven pairs of primers were designed to amplify and sequence the sixteen HPA systems. The HPA genotypes of two standard samples were laa/2aa/3ab/4aa/5ab/6aa/7aa/8aa/9aa/10aa/11aa/12aa/13aa/14aa/15aa/16aa and laa/ 2aa/3aa/4aa/5aa/6aa/7aa/8aa/9aa/10aa/11aa/12aa/13aa/14aa/15aa/16aa, respectively. The 256 HPA genotypes of 16 reference samples were clear. 128 genotypes among them were completely accordance with the results provided by ISBT report. Conclusions The PCR-SBT assay combining high-throughput DNA sequencer established in the study provides a simple, rapid and accurate method for HPA-1 to HPA-16w systems genotyping. The assay is suitable for routine clinical HPA genotyping and shows a broad prospect in further applications.