应用PCR-SSP方法进行人类血小板抗原1～6系统的基因分型

Study on the genotyping of human platelet antigens of 1,2,3,4,5,6 system by PCR-SSP

目的研究采用PCR-SSP技术,建立人类血小板抗原1～6系统(HPA-1,2,3,4,5,6)的基因分型方法。方法合成18条序列特异性引物,通过调节引物浓度、Mg2+离子浓度和探索最佳PCR扩增条件,建立HPA-1～6系统同步基因分型技术。对第10届及第11届国际输血协会(ISBT)血小板基因定型协作组送检的考核样本进行盲检来验证。并应用该技术对198名深圳地区健康的血小板志愿捐献者进行基因分型。结果应用本研究的方法,对第10届及第11届ISBT送检的考核样本进行基因分型,结果与ISBT公布的结果完全一致,符合率达100%。对198名随机的血小板志愿捐献者观察到的基因频率分别是:HPA-1a和1b为0.9924和0.0076,HPA-2a和2b为0.9545和0.0455,HPA-3a和3b为0.5556和0.4444,HPA-4a和4b为0.9975和0.0025,HPA-5a和5b为0.9848和0.0152,HPA-6a和6b为0.9798和0.0202。结论本研究建立的HPA基因分型技术具有简便、快速、准确的特点,适合于常规HPA基因分型,具有广泛的应用前景。

Objective To set up the reliable genotyping of human platelet antigens （HPA） of 1,2,3,4,5,6 system by PCR-SSP. Methods In this study, 18 sequence-specific primers were designed and synthesized. The concentration of each primer pair, the concentration of Mg^2＋ and the PCR conditions were adjusted to the optimum so that HPA-1 to 6 systems could be amplified simultaneously under the same PCR cycling parameters. The accuracy and reliability of the assay evaluated was evaluated and confirmed by typing the coded DNA samples distributed by the 10TM and 11TM Platelet Genotyping Workshop of the International Society of Blood Transfusion （ISBT）. A total of 198 volunteer platelet donors in Shenzhen wrere genotyped at HPA-1 to HPA-6 systems. Results The coded samples distributed by the 10TM and 11TM Platelet Genotyping Workshop of ISBT were genotyped by PCR-SSP method, a concordance rate of 100 percent was observed between our results and the results provided by ISBT report. The HPA gene frequencies observed in 198 randomly-selected healthy platelet domors in Shen zhen were 0.9924 and 0.0076 for HPA-1a and lb, 0.9545 and 0.0455 for HPA-2a and 2b, 0.5556 and 0.4444 for HPA-3a and 3b, 0.9975 and 0.0025 for HPA-4a and 4b, 0.9848 and 0.0152 for HPA-5a and 5b, 0.9798 and 0.0202 for HPA-6a and 6b respectively. Conclusions PCR-SSP assay provided a simple, rapid and accurate method for HPA genotyping, it can be used in routine clinical HPA genotyping and shows a broad prospect in its further applications.