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目的通过对PCR-DNA技术的分析,探讨人类白细胞抗原(HLA)基因分型方法。方法采用PCR反向序列特异性寡核苷酸(polymerase chain reaction-reverse sequence specific oligonucleotide,PCR-RSSO)杂交技术,建立改良半量扩增体系全自动HLA-I、Ⅱ等位基因分型方法,进行了635份血液标本HLA-A、B、C、DR、DQ等位基因分型,其中166份DNA同时采用序列特异性引物技术(PCR-SSP)和手工全量扩增体系PCR-RSSO技术。对全自动半量PCR-RSSO、PCR-SSP、手工PCR-RSSO 3种方法做两两比较。结果全自动半量PCR-RSSO的分型成功率为98.4%(3 124/3 175),PCR-SSP为98.8%(656/664),手工PCR-RSSO为88.3%(733/830)。经χ2检验,全自动半量PCR-RSSO与PCR-SSP的分型成功率无统计学差异,与手工PCR-RSSO有显著差异(P<0.05)。结论PCR-RSSO可识别HLA-Ⅰ,Ⅱ共706个等位基因,覆盖WHO命名委员会2000年公布的936个等位基因的75.43%;对706个HLA等位基因的分型均为中~高分辨率,有分辨纯合子等位基因的能力;易长期保存书面的实验原始资料,即杂交条;具有成本低、劳动强度低、省时和DNA消耗量少等优点。PCR-RSSO适合于造血干细胞移植和建立造血干细胞及脐带血干细胞库的组织配型。
Objective To investigate the genotyping of human leukocyte antigen (HLA) by PCR-DNA technology. Methods The PCR-RSSO hybridization technique was used to establish the automatic HLA-I and HLA-Ⅱ allelic genotyping methods for improved semi-quantitative amplification system. The HLA-A, B, C, DR and DQ alleles of 635 blood samples were genotyped, of which 166 DNAs were sequenced by PCR-SSP and PCR-RSSO. The comparison of the three methods of automatic semi-quantitative PCR-RSSO, PCR-SSP and manual PCR-RSSO was done. Results The success rate of semi-quantitative PCR-RSSO typing was 98.4% (3 124/3 175), PCR-SSP was 98.8% (656/664) and manual PCR-RSSO was 88.3% (733/830). After χ2 test, there was no significant difference in the success rate of semi-quantitative PCR-RSSO and PCR-SSP, which was significantly different from that of manual PCR-RSSO (P <0.05). Conclusion PCR-RSSO can identify 706 alleles of HLA-Ⅰ and Ⅱ, covering 75.43% of the 936 alleles published by WHO Nomenclature Committee in 2000. The typing of 706 HLA alleles was moderate to high Resolution, ability to distinguish homozygous alleles; long-term preservation of written original experimental data, that is, hybridization; with low cost, low labor intensity, time saving and DNA consumption less advantages. PCR-RSSO is suitable for hematopoietic stem cell transplantation and establishment of histological configurations of hematopoietic stem cells and cord blood stem cell pools.