序列特异引物聚合酶链反应在HLA-B27检测中的应用

Application of polymerase chain reaction using sequence-specific primers in detection of HLA-B27

目的探讨序列特异引物聚合酶链反应(PCR-SSP)方法在人类白细胞抗原B27检测中的应用。方法对经流式细胞术分析定型的279例疑似强直性脊柱炎患者样本采用PCR-SSP法检测HLA-B27的表达。以流式细胞术检测结果为判断标准,对结果不一致样本,进一步应用PCR-直接测序法进行B27测序分析。结果与流式细胞术结果相比,只有1例结果不一致。经测序分析,其碱基序列与PCR-SSP法特异性引物3′末端错配;且发现该样本序列存在多个杂合碱基,经与HLA-B27等位基因数据库比对,该杂合序列与最相近B*2733亚型存在2个碱基差异。结论 PCR-SSP法可适用于HLA-B27的临床检测。

Objective To investigate the effect of polymerase chain reaction using sequence-specific primers（PCR-SSP） in detecting Human leukocyte antigen B27（HLA-B27）.Methods Peripheral blood samples from 279 cases of suspected patients with ankylosing spondylitis（AS） were collected,which had been identified by flow cytometry（FCM）.Genomic DNA was analyzed by PCR-SSP.Comparing with the results of flow cytometry,the discrepancies were then analyzed by the sequence-based typing method.Results Except for one sample,the results of PCR-SSP were in agreement with flow cytometry.The sequencing results of the discrepancy showed its nucleotide sequence mismatch with the 3′end of the specific primer and several heterozygous nucleotides in exon 2 and 3.In alignment with HLA Alleles database,there are 2 nucleotide changes from B＊2733 in heterozygous sequences.Conclusion The PCR-SSP technique is applicable for the clinical detection of HLA-B27.