ABSTRACT
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 d 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 1aa/2aa/3ab/4aa/5ab/6aa/7aa/8aa/9aa/10aa/11aa/12aa/13aa/14aa/15aa/16aa and 1aa/ 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.