高分辨率熔解曲线用于RhCE血型基因分型的研究

RhCE blood group genotyping by high-resolution melting curve

目的探讨高分辨率熔解曲线(HRM)用于RhCE血型基因分型的可行性。方法随机选取无输血史患者300例, 进行RhCE血型血清学表型分型, 从中选出表型为CCee, CcEe, ccEE的患者标本各10例, 共30例;表型CCee的为1组, 表型CcEe的为2组, 表型ccEE的为3组, 互为对照组, 提取基因组DNA, 设计引物3对, 利用HRM基因分型方法对RhCE血型进行基因分型实验。结果 30例标本的HRM基因分型结果与血清学表型一致, 引物对1的HRM可将各组标本中基因型为CC的检出;引物对2的HRM可将各组标本中CC, Cc, cc基因型全部检出;引物对3的HRM可将各组标本中基因型为Ee的检出;在检测过HRM的引物对3实验产物中加入1组单纯PCR扩增产物后再次行HRM检测, 可将各组标本中EE, Ee, ee基因型全部检出。结论高分辨率溶解曲线可用于RhCE血型基因分型。

Objective The purpose of this study was to investigate the feasibility of RhCE blood group genotyping by high-resolution melting curve(HRM)analysis.Methods A total of 300 transfusion-naïve patients were randomly included for serological RhCE phenotyping.From these,30 samples comprising 10 each with phenotypes CCee(group 1),CcEe(group 2),and ccEE(group 3)were selected.The three groups were set as controls between one another.Genomic DNA was extracted,3 primer pairs were designed,and then the RhCE blood group was further determined by HRM genotyping.Results The HRM genotyping results of these 30 samples were in accordance with the phenotypes by serology.In HRM analysis,the primer pair 1 identified all samples with genotype CC primer pair 2 identified all samples with genotypes CC,Cc,and cc,and primer pair 3 identified all samples with genotypes Ee,in each group.After the PCR amplification products of group 1 was added to the experimental products of primer pair 3,a second HRM analysis identified all samples with genotypes EE,Ee,and ee in each group.Conclusion Therefore,high-resolution melting curves can be used for genotyping of RhCE blood groups.