PCR-RSSO基础上HLA-Ⅰ、Ⅱ基因分型的研究

HLA genotyping by automatic semi-quantitative polymerase chain reaction-reverse sequence specific oligonucleotide

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％（3124／3175），PCR－SSP为98．8％（656／664），手工PCR－RSSO为88．3％（733／830）。经χ^2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36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 explore a HLA genotyping method that can be used for organ transplantation tissue typing, and especially for establishing hemopoietic stem cell bank and the cord blood stem cell bank by analyzing the PCR-DNA. Methods Modified automatic method based on semi-quantitative amplification system of HLA-Ⅰ,Ⅱallele genotyping was established using reverse sequence-specific oligonucleotide with polymerase chain reaction (PCR-RSSO), and was compared with PCR (using sequence specific primer, PCR-SSR) and manual PCR-RSSO in terms of the accuracy, resolution, and the quantity of DNA consumption. A total of 635 blood samples were genotyped with HLA-A, B, C, DR and DQ alleles using auto semi-quantitative PCR-RSSO, 166 of which were also examined using PCR-SSP and manual PCR-RSSO simultaneously. Results The success rate of automatic semi-quantitative PCR-RSSO, PCR-SSP and manual PCR-RSSO was 98.4% (3 124/3 175), 98.8% (656/664) and 88.3% (732/830) respectively, with no significant difference between the former 2 as indicated by χ 2 test (P>0.05). Auto semi-quantitative PCR-RSSO, however, yielded significantly higher success rate than manual PCR-RSSO (P<0.05). Conclusion PCR-RSSO is capable of identifying 706 alleles of HLA-I, II antigens which amounts to 75.43% of the 936 alleles published by WHO in 2000, with intermediate to high resolution, even in the case of the homozygote. The hybridization results documenting the original data can be conveniently preserved. This method therefore possesses the merits of low expenses, low labor intensity, and rapid processing of large number of samples.