中国人特异性的RHD基因定型方法的建立

RHD genotyping for the Chinese

目的 建立特异性针对中国人的RHD基因定型方法。方法 设计6对特异性针对中国人RHD等位基 因的引物,并引入1对内对照引物,建立PCR 序列特异性引物(PCR- SSP)方法,分6个反应管检测RHD基因。采 用这一方法检测经血清学鉴定和RHD全基因序列分析的88份中国汉族人Rh阴性样本,13份Rh阳性、弱D型和 部分D表型样本,以及28份D放散型(Del)样本,同时对318名随机无偿献血者作RHD基因定型,然后与血清学检 测结果比较。结果 所有样本的PCR SSP检测结果均与血清学结果一致(符合率100%),并可指示目前在中国人 中观察到的全部RHD基因阳性、D抗原阴性等位基因(或称无效等位基因),同时还可检出Del表型及存在于Rh阳 性个体中的Del等位基因(RHD1227A)即RHD/Del杂合型(或RHD/RHD1227A);对318名随机个体的RHD 基因检测显示后者在中国人群中的比例为8/318,Del基因在中国人群中频率即为0.012579。结论 上述PCR-SSP 方法简便、快速,且具有较高的准确率,可用于临床或科研进行中国人RHD基因定型或遗传分析。

Objective To establish a RHD genotyping method specific for the Chinese. Methods Six pairs of primers specific for most alleles found in the Chinese according to the records in NCBI GenBank, were designed, and a multi-tube sequence-specific primer PCR (PCR-SSP) method was established with a pair of internal control primer in each reaction. The method was evaluated with samples serologically determined and full length RHD sequenced from 89 Rh-negative, 28 D el, and 13 Rh-positive, weak D and partial D phenotype of Chinese Hans. Furthermore, 318 random samples from blood donors were genotyped and the results were compared with serological results of those samples. Results The PCR-SSP results were in concordance with serological results (100%) in all samples, and all RHD positive, D antigen negative alleles (or nonfunctional alleles) observed in the Chinese up to now could be detected or implicated, including D el phenotype especially D el allele existing in Rh-positive individuals (RHD/RHD1227A). This genotype was detected with a rate of 8/318, and allele frequency should be 0.012579 Conclusion Our method is rapid and easy, with high accuracy in the testing of the Chinese.