参照链介导的构象分析策略在HLA-B位点分型中的临床应用研究

HLA-B locus genotyping with reference strand mediated conformation analysis (RSCA) system in clinical usage

目的建立并稳定HLAB位点的参照链介导的构象分析(RSCA)系统,并鉴定RSCA分型策略的优势。方法提取27例造血干细胞移植患者及63名供者的外周血DNA,采用RSCA进行HLAB位点分型,并与序列特异性引物聚合酶链反应技术(PCRSSP)分型结果进行比较,对于疑难标本采用测序鉴定。结果在90份标本中,87份(96.7%)可明确判定结果,其中有1份标本与PCRSSP结果不一致,经测序鉴定RSCA结果正确;67份(74.4%)可直接指定等位基因型别。此外,1份标本(1.1%)因PCR扩增结果太弱而未能获得RSCA分型结果,2份标本只能确定1条等位基因,经测序发现另1条等位基因为已知等位基因,但RSCA数据库中没有其数据。随机选择20份标本进行RSCA重复性实验,重复率为100%。在PCRSSP方法分型中,约有10%的标本需要重复1～3次方能判定结果。结论RSCA具有灵敏、特异、分辨率高、重复性好、可发现变异基因及新的等位基因、大规模和低成本等优点,适用于大规模的无关移植供、受者的选择。

Objective To establish and stabilize a new HLA-B locus typing strategy, reference strand mediated conformation analysis(RSCA)system and to conform its advantages.Methods Of 27 hematopoitic stem cell transplant patients and 63 potential donors DNA samples were extracted from peripheral blood cells and HLA-B loci were both typed by RSCA and PCR-SSP methods. The (ambi)-(guous) results were identified by using DNA sequencing. Results Among 90 samples, 87/90 ((96.7) %) cases could be designated definitely, but one of them was disagreement with the SSP result which was confirmed by sequencing, and 67/90 ((74.4) %) cases could be typed to allelic level. 1/90 ((1.1) %) case could not be identified by RSCA for its bad PCR results. Only one allele of each 2 samples could be designated and the other could not be identified by RSCA, which were further confirmed by sequencing method, and the results confessed which were known as alleles, but there were no corresponding data in RSCA database. Twenty samples were randomly selected to identify the replication rate of RSCA, and the results demonstrated that the replication rate was 100 %. Among PCR-SSP typing results, about 10 % samples might be typed for 1-3 times to confirm their types. Conclusion RSCA had some advantages such as high resolution, high sensitivity, high accuracy, high replication, finding new alleles, large scale and low cost, and was especially suitable for unrelated donor-recipient (screening).