两步法分析RhD阴性及D变异体分子机制(附1例新等位基因的发现)

Two-step approach for RhD gene analysis of of RhD negatives and variants and the discovery of a new allele

目的建立用于RhD阴性及RhD变异体大标本量的快速准确的筛查方法。方法采用血清学常规试管法初筛后，对初筛阴性标本以序列特异性引物多聚酶链反应（SSP—PCR）作两步法（第一步检测出阴性，D^el1227突变，以及其他变异体；第二步检定出其他变异体的具体类型）基因检测分型，并用INNO—TRAIN试剂盒作检测结果的比较，对有突变位点的变异体进行基因测序。结果在235例初筛RhD阴性标本中，检测到1—10外显子全缺失137例，2～9外显子缺失20例，1227G〉A突变63例，845G〉A突变4例，3～6外显子缺失2例，3G〉A突变1例，8～9外显子缺失1例，首次发现1例3—10外显子缺失；INNO—TRAIN试剂检测及基因测序结果与之完全符合。结论所建立的两步法基因检测分型，方法经济实用、简洁可靠，适用于大标本量筛查RhD阴性及D变异体。

Objective To establish a tow-step approach for genotyping RhD negative blood samples indentified by serological tests, which could help blood centers detecting real RhD negative and RhD variants exactly and rapidly. Methods Genomic DNA was extracted from the RhD negative specimens indentified by serological testing. RHD genotyping was performed by using polymerase chain reaction with sequence-specific primers （SSPPCR） of our kits and INNO-TRAIN kits, and the samples with point mutations were sequenced. Results Among 235 RhD negative samples, there were 137 samples with deletion of 1 - 10 exons,20 with deletion of 2 -9 exons,63 with 1227G 〉 A mutation,4 with 845G 〉 A mutation,2 with deletion of 3 - 6 exons, 1 with 3G 〉 A mutation, 1 with deletion of 8 - 9 exons, and 1 with deletion of 3 - 10 exons, which was reported for the first time. The results were exactly the same, when compared to INNO-TRAIN reagent testing and gene sequencing. Conclusion The two-step approach established in this study could be used for screening real RhD negatives and RhD variants. This new approach is simple and cost-effective.