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多重PCR-SSP技术应用于HPA-1~17bw基因分型研究和广西壮族人群HPA多态性的分布
(来源:南宁输血医学研究所 )
作者:申卫东  李丽兰   廖燕  赵桐茂  吴国光来源:中国输血杂志, 2014, 27(1):8-14. 摘要:目的   建立应用于HPA-1~17bw基因分型的多重聚合酶链反应-序列特异性引物( PCR-SSP )技术,对HPA多态性分布进行调查。 方法  设计多种HPA序列特异性引物的组合,在1个PCR反应体系中同时扩增多个HPA基因,根据扩增片断的有无和大小,指定相应的HPA基因型,并以17例国际合作研究项目提供的HPA参比DNA配组的标本,以及100例已知HPA型的血小板供者的DNA标本作验证。使用验证后的技术对2 659名无血缘关系的广西壮族人群展开HPA-1~17bw的基因分型和抗原多态性研究。 结果 建立了由16种PCR引物混合液,其中有6种多重PCR引物混合液组成的PCR-SSP反应体系,可用于HPA-1~17bw等位基因的检测。以HPA参比DNA配组标本,以及已知HPA型的供者的DNA标本的检测作验证,HPA分型结果完全一致;在2 659名广西壮族人群中各HPA等位基因频率分布为:HPA-1a和-1b为0.991 5和0.008 5,HPA-2a 和 -2b为 0.956 9和 0.043 1,HPA-3a 和 -3b为 0.512 2和 0.487 8;HPA-5a 和 -5b为0.985 0和0.015 0,HPA-6a 和 -6b为 0.985 1和0.014 9,HPA-15a和-15b为0.525 6和0.474 4;HPA-4、HPA-7~14bw、HPA-16~17bw只有a/a的纯合子;HPA-2~3、HPA-6bw以及HPA-15均存在a/a和b/b纯合子基因型个体。 结论 多重PCR-SSP 技术应用于HPA基因分型减少了PCR扩增反应数量,节省了人力、物力,HPA分型系统性强,可用于大量标本的HPA-1~17bw基因分型等研究和筛查工作;应用该方法研究证明广西壮族人群HPA多态性分布具有民族特征,有助于对壮族人群血小板免疫学特点的了解。    关键词:人类血小板特异性抗原(HPA);HPA基因分型;多重PCR-SSP;壮族;广西The Study on Multi-PCR-SSP Technique for HPA-1~17bw Genotyping and the Distribution of HPAs Polymorphism in Chinese Zhuang PopulationWei-Dong SHEN,  Li-Lan LI,  Yan LIAO,  Tong-Mao ZHAO,  Guo-Guang WU.  Chinese Journal of Blood Transfusion, 2014, 27(1):8-14.Abstract:  Objective   To develop a multi-PCR-SSP technique for human platelet antigens (HPAs) -1 to -17bw genotyping. And study on the distribution of HPAs polymorphism in Zhuang population by this technique. Methods   Designed various combinations of HPA sequence-specific primers in order to simultaneously amplify several target HPA gene fragments in one PCR reaction by using one multi-primers combination. The results of HPA genotype were read and assigned according to the size of the target fragment and the presence or absence of the fragment. The effectiveness of the multi-PCR-SSP technique was evaluated by using 17 HPA reference DNA samples that were provided in 14th Platelet Immunology Workshop and 100 HPA-known DNA samples of platelet donors. Total of 2 659 random samples from unrelated and healthy adults of Zhuang population in Guangxi region were studied for HPA -1 to -17bw genotype and antigens polymorphism by the developed technique.  Results   There are 16 primers mixes in the multi-PCR-SSP method, and 6 of them are multi-PCR primers combinations mixes in our technical system. 100% concordance of genotyping results were showed by the evaluation of using 17 reference DNA samples and 100 DNA samples of known-HPA-genotype platelet donors. The HPAs allele frequencies in Zhuang population were 0.991 5 and 0.008 5 for HPA-1a and -1b; 0.956 9 and 0.043 1 for HPA-2a and -2b; 0.512 2 and 0.487 8 for HPA-3a and -3b; 0.985 0 and 0.015 0 for HPA-5a and -5b; 0.985 1 and 0.014 9 for HPA-6a and -6b; 0.525 6 and 0.474 4 for HPA-15a and -15b, respectively. There only a/a homozygosis was detected in HPA-4, HPA-7~14bw, -16~21bw. And a/a and b/b homozygosis in HPA-2~3, HPA-6bw and HPA-15 were found.   Conclusion   In this study, we successfully established a multi-PCR-SSP technique for HPA-1 to -17bw genotyping. It was a rapid and accurate method for HPA genotyping, particularly suitable for large quantity samples’ HPA-1~17bw genotyping. This study showed that the HPAs polymorphism in Zhuang population had their own national characteristics, and the data helps us to know the characteristics of platelet immunological in Zhuang population.Key Words: Human Platelet Antigen (HPA); HPA Genotyping; Multi-PCR-SSP; Zhuang population; Guangxi; 



时间: 2015 年 09 月 15 日
           
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