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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,对广西壮族人群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2659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。
Objective To establish a multiplex polymerase chain reaction-sequence-specific primer (PCR-SSP) technique for genotyping HPA-1 to 17bw and investigate the distribution of HPA polymorphism in Guangxi Zhuang population. Methods A combination of multiple HPA sequence-specific primers was designed to amplify multiple HPA genes in one PCR reaction system. According to the presence and absence of amplified fragments, the corresponding HPA genotypes were assigned and 17 cases of international cooperation The HPA reference DNA kit provided by the research project, together with DNA samples from 100 platelet donors of known HPA type, were validated. Using validated techniques, genotyping and antigenic polymorphism of HPA-1 ~ 17bw were performed in 2659 non-related Guangxi Zhuang nationality. Results The PCR-SSP system consisted of sixteen kinds of PCR primers and six kinds of multiplex PCR primers were used to detect the HPA-1 ~ 17bw allele. The results of HPA typing were consistent with the detection of HPA reference DNA and DNA samples of known HPA donors. The frequencies of HPA alleles in 2 659 Guangxi Zhuang populations were: HPA -1a and -1b were 0.991 5 and 0.008 5, HPA-2a and -2b were 0.956 9 and 0.043 1, HPA-3a and -3b were 0.512 2 and 0.487 8 respectively, HPA-5a and -5b were 0.985 0 and 0.015 0 , HPA-6a and -6b were 0.985 1 and 0.014 9, HPA-15a and -15b were 0.525 6 and 0.474 4 respectively; only a / a homozygotes for HPA-4, HPA-7-14bw and HPA-16-17bw; HPA-2 ~ 3, HPA-6bw and HPA-15 exist a / a and b / b homozygous genotype individuals. Conclusion The application of multiplex PCR-SSP technology in HPA genotyping reduces the number of PCR amplification reactions, saves manpower and material resources, and has a strong systematic typing of HPA. It can be used for genotyping of HPA-1 ~ 17bw in a large number of samples, Investigation work; application of this method to study the distribution of HPA polymorphism in Zhuang nationality in Guangxi Zhuang nationality characteristics, contribute to the knowledge of platelet immunology characteristics of Zhuang people.