使用Taqman PCR技术建立人类血小板抗原-1～5、15分型体系

Establishment of HPA-1-to-5 and HPA-15 genotyping systems by Taqman PCR

目的了解上海地区单采血小板献血人群HPA-1～5、15多态性分布,分析评估新的分型技术。方法利用TaqMan PCR技术对500份上海地区单采血小板供者标本进行HPA-1～5、15抗原系统等位基因分型,并随机抽取100份标本使用PCR-SSP技术进行比对。结果 HPA各等位基因频率分别为HPA-1a:0.999,HPA-1b:0.001,HPA-2a:0.953,HPA-2b:0.047,HPA-3a:0.582,HPA-3b:0.418,HPA-4a:0.999,HPA-4b:0.001,HPA-5a:0.988,HPA-5b:0.012,HPA-15a:0.524,HPA-15b:0.476;有1份标本HPA-5等位基因与SSP检测结果产生差异。结论上海地区HPA各等位基因频率与国内各地区人群分布无明显差异,与中国汉族人群HPA分布情况基本吻合,实验数据经验证符合Hardy-Weinberg平衡定律,实验结果准确可靠;HPA-5差异经测序验证判断可能由PCR-SSP非特异扩增所致;TaqMan技术在HPA抗原系统分型应用中特异性高,反应时间短,具有良好的应用前景,是现有技术方法的一种重要补充。

Objective To investigate gene frequencies of HPA-1-to-5 and HPA-15 from apberesis platelet donors in Shanghai,and evaluate a new genotyping technique. Methods A total of 500 platelet aphresis donors were genotyped for HPA-1-to-5 and HPA-15 antigen systems by means of Taqman PCR, and 100 samples were selected randomly for comparison by PCR-SSP. Results The gene frequencies of HPA-la,HPA-1b,HPA-2a,HPA-2b,HPA-3a,HPA-3b,HPA-4a,HPA-4b, HPA-Sa,HPA-Sb ,HPA-15a and HPA-15b identified using Taqman PCR were 0. 999 ,0. 001,0. 953 ,0. 047,0.582,0.418, 0.999,0.001,0.988,0.012,0. 524 and 0. 476, respectively. In one case for typing HPA-5 allele, the result obtained by Taqman PCR was different compared with PCR-SSP. Conclusion The allele gene frequencies of HPA systems are not significantly different between Shanghai area and other regions in China, meanwhile, the results are comparable to the frequency distribution of HPA in Chinese Hans and fit Hardy-Weinberg equilibrium. Difference observed in the distribution of HPA-5 may be the result of nonspecific amplification of PCR-SSP according to sequencing confirmation. Taqman PCR technique with high specificity and timesaving advantages has good application prospect in typing for HPA systems,which is available as a significant complement to existing methods.