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目的建立HPA-1-28w基因分型检测技术体系并用以研究广西瑶族和汉族人群的血小板抗原(HPA)-1-28w基因多态性。方法通过设计序列特异性引物和优化PCR反应条件,摸索HPA-1-28w基因分型检测技术体系,其组成包括前期已建立的HPA-1-17w多重PCR-SSP技术和新研发的HPA-18-28w PCR-SSP方法 2部分组成。使用10例已预先测序确定HPA-18-28w基因型和20例已预先使用商品试剂盒检测HPA-18-21w基因型的本研究广西瑶族标本,对新研发的HPA-18-28w PCR-SSP方法做验证。应用所建立的HPA-1-28w基因检测技术对广西地区无血缘关系的1424名健康瑶族人和908名健康汉族人做HPA-1-28w基因分型和多态性分析。结果所建立的HPA-1-28w基因分型技术系统,不仅对HPA-18-28w基因的分型结果与对验证用标本的测序分型结果完全一致,而且对HPA-18-21w基因的分型检测结果与商品试剂盒的分型结果完全一致。广西瑶族人群的HPA各等位基因频率分别为:HPA-1a 0.998 2、1b 0.001 8,2a 0.874 6、2b 0.125 4,3a 0.661 5、3b 0.338 5,5a 0.996 8、5b 0.003 2,6a 0.9786、6bw 0.021 4,15a 0.407 7、15b 0.592 3;汉族人群HPA各等位基因频率分别为:HPA-1a 0.998 3、1b 0.001 7,2a0.957 6、2b 0.042 4,3a 0.532 5、3b 0.467 5,4a 0.999 4、4b 0.000 6,5a 0.984 0、5b 0.016 0,6a 0.989 0、6bw 0.011 0,15a 0.534 1、15b 0.465 9;广西瑶族和汉族人群的HPA-7-14w、HPA-16-28w基因均以aa纯合子形式存在。在本组广西瑶族人群中发现2例罕见的HPA-6bb纯合子基因型个体,而在汉族人群中发现了1例中国人群中罕见的HPA-4ab基因型个体。结论建立了完善的HPA-1-28w PCR-SSP基因分型检测技术体系,并成功应用于广西瑶族和汉族人群的HPA-1-28w基因筛查和分型研究;广西瑶族、汉族人群HPA基因频率分布在HPA-2,-3,-5,-15系统和HPA-6w等均不同(P<0.05),藉此有助于了解广西瑶族和汉族人群HPA的免疫学特点。
OBJECTIVE: To establish a HPA-1-28w genotyping assay system and to study the genetic polymorphisms of platelet antigen (HPA) -1-28w in Guangxi Yao and Han populations. Methods By designing sequence-specific primers and optimizing PCR reaction conditions, HPA-1-28w genotyping detection system was explored, which consisted of HPA-1-17w multiplex PCR-SSP technology and newly developed HPA-18 -28w PCR-SSP method 2 parts. Ten cases of HPA-18-28w genotype have been pre-sequenced and 20 cases of HPA-18-21w genotypes have been pre-tested using commercial kits in this study. The newly developed HPA-18-28w PCR-SSP Method to verify. The established HPA-1-28w gene detection technique was used to analyze the genotypes and polymorphisms of HPA-1-28w in 1424 healthy Yao nationality and 908 healthy Han nationality in Guangxi province. Results The established HPA-1-28w genotyping system not only completely matched the typing results of the HPA-18-28w gene with the sequencing results of the samples used for validation, but also analyzed the distribution of the HPA-18-21w gene Type test results and commercial kit typesetting results are exactly the same. The prevalences of HPA alleles in Yao population of Guangxi were: HPA-1a 0.998 2,1b 0.001 8,2a 0.874 6,2b 0.125 4,3a 0.661 5,3b 0.338 5,5a 0.996 8,5b 0.003 2,6a 0.9786, 6bw 0.021 4,15a 0.407 7,15b 0.592 3; Han Chinese population HPA allele frequencies were: HPA-1a 0.998 3,1b 0.001 7,2 a0.957 6,2 b 0.042 4,3a 0.532 5,3b 0.467 5, 4a 0.999 4,4b 0.000 6,5a 0.984 0,5b 0.016 0,6a 0.989 0,6bw 0.011 0,15a 0.534 1,15b 0.465 9; HPA-7-14w, HPA-16-28w genes in Guangxi Yao and Han populations All exist as aa homozygotes. Two cases of homozygous HPA-6bb genotypes were found in this group of Yao nationality in Guangxi, and one case of HPA-4ab genotype rare in Chinese population was found in Han population. Conclusion The established HPA-1-28w PCR-SSP genotyping detection system was established and successfully applied to the screening and genotyping of HPA-1-28w gene in Guangxi Yao and Han populations. The HPA-1-28w The frequency distribution was different between HPA-2, -3, -5, -15 and HPA-6w (P <0.05), which could help to understand the immunological characteristics of HPA in Guangxi Yao and Han populations.