血型基因检测对照品的制备及其在三种稀有血型筛选中的应用

目的 应用基于聚合酶链反应(polymerase chain reaction,PCR)的基因定点诱变技术(sitedirected mutagenesis,SDM)制备s、Oka血型等位基因检测对照品.用多重聚合酶链反应(multiplex PCR)技术建立3种血型Fy2、s和Ok2的基因分型方法,以了解这3种血型在中国随机献血者中的多态性分布状况.方法 采用基于PCR的基因定点诱变技术对s和Oka血型等位基因的单核苷酸多态性(single nucleotide polymorphism,SNP)位点构建了含有突变SNP位点(GYPB基因cDNA 153位C/T突变和BSG基因cDNA 274位G/A突变)的标准质粒,作为s和Oka血型等位基因检测的对照品.同时针对血型抗原Fya、s和Oka等位基因的SNP位点设计序列特异性(sequence specific primer,SSP)引物,构建多重PCR体系,对438份随机献血者样本进行Fya、s和Oka血型抗原的基因筛选.结果 成功应用定点诱变技术完成了s和Oka基因检测中等位基因对照品的制备,并建立了可同时检测Fya、s和Oka血型的多重PCR体系,438份随机献血者样本中共检出2例Fy(a-)样本,未检出s-和Ok(a-)样本.结论 基于PCR的基因定点诱变技术能够得到难以获得的血型基因检测等位基因对照品,用于验证基因分型方法.本实验建立的多重PCR体系是一种有效的进行Fya、s和Oka血型基因检测的方法.

The establishment of the controls for blood group genotyping and the application in the screening of three rare blood groups

Objective To establish the controls for allele detection of blood groups s and Oka. A multiplex PCR method for the detection of three blood group antigens Fya, s and Oka was developed and used to investigate the distribution of these blood groups in Chinese random blood donors. Methods Polymerase chain reaction (PCR)-based, gene site-directed mutagenesis (SDM) technique were used to make site-directed mutagenesis for the single nucleotide polymorphism (SNP) sites of the blood group alleles (the 153 C/T point mutation of the GYPB gene, and the 274 G/A point mutation of the BSG gene) as controls for allele detection. Sequence specific primers were designed according to the SNP sites of alleles of blood group antigens Fya, s and Oka. A multiplex PCR system was developed and 438 random donor samples were screened for the blood group antigens Fya, s and Oka. Results The controls for alleles in blood groups s and Oka were successfully made with the SDM technique, a multiplex PCR system was set up and successfully used to analyze the genotypes of three blood group antigens Fya, s and Oka. Two Fy(a-)samples were detected in the 438 samples, no s- and Ok(a-) sample was found. Conclusion The PCR-based SDM technique can be used to obtain the unavailable controls in blood group genotyping. The multiplex PCR technique established in this study is an efficient genotyping method for blood groups Fya, s and Oka.