两种不同的KIR基因分型方法的对比研究

Comparative Study on the Typing of Killer Immunoglobulin-Like Receptor by Using Two Different Genotyping Methods

目的探讨常用的序列特异性引物-PCR（PCR—SSP）及流式序列特异性寡核苷酸探针（Flow-rSSO）杂交方法在杀伤细胞免疫球蛋白样受体（KIR）框架基因分型结果中的符合率和准确性。方法随机选择2011年6月77例深圳造血干细胞捐献者的外周血样，分别用PCR-ssp及Flow-rSSO商品化试剂盒进行KIR框架基因定型，分别通过凝胶电泳图及HLA Fusion 2．0 Research软件分析法，分析结果的一致性。对初检结果不一致的样本，采用同一厂家、不同批号的商品化试剂盒进行复检，并采用美国国立卫生研究院癌症研究所KIR PCR-SSP方法进行检测。结果77例样本中，75例完全一致，另2例（2．6％）KIR 2DL2的结果不一致，PCR—SSP方法对KIR 2DL2的初检、复检检测结果均为阴性，但用Flow-rSSO Lot#004批号试剂盒检测结果为阳性。更换新的Flow—rSSO Lot#005批号试剂盒复检，结果与PCR—SSP方法的检测结果相一致。结论为保证KIR基因分型的准确性，开展KIR基因分型的室内、室间质控工作至关重要。

Objective To explore the accuraey of killer immunoglobulin-like receptor （KIR） genotyping by using the sequence specific primer-PCR （PCR-SSP）and flow reverse sequence specific oligonucleotide probe（Flow-rSSO）assays. Methods A total of 77 samples from randomly-selected stem cell volunteer donors in Shenzhen were subjected to KIR genotyping using the commercial PCR-SSP and Flow- rSSO kits, respectively. The accordance of KIR genotyping results was evaluated and the reason causing the different results was analyzed in this study. Samples with inconclusive and different genotyping results were retested using the same PCR-SSP and Flow-rSSO commercial kits with updated lot number and also typed by PCR-SSP method established by National Cancer Institute. Results In the 77 tested samples, the KIR genotypes of 75 samples were in accordance by PCR-SSP and Flow-rSSO commercial kit. However, the other two samples showed different results at KIR2DL2 frame gene： the commercial and NCI PCR-SSP genotyping results indicated the KIR2DL2 were absent, whereas KIR2DL2 were defined present by Flow- rSSO using the initial Lot # 004 commercial kit. We used the updated Flow-rSSO Lot # 005 commercial kit, KIR2DL2 were demonstrated to be absent for the two samples, which is in accordance with results typed by PCR-SSP. Conclusions This study implied that the internal and external quality control would be critical important and essential in KIR genotyping.