Department of Clinical Chemistry, Diakonessenhuis, Utrecht, The Netherlands.
Clin Chem Lab Med. 2011 Sep 6;49(12):1979-85. doi: 10.1515/CCLM.2011.252.
Human leukocyte antigen B27 (HLA-B27) is strongly associated with ankylosing spondylitis. The B27 allele is present in 90% of patients with this disease, whereas it is present in only 9% of Caucasians. Molecular detection of HLA-B27 is traditionally based on allele specific amplification of exon 2 (Olerup method) or exon 3 (Dominguez method) by PCR, followed by gel analysis.
We developed a real-time TaqMan PCR based on the Dominguez method with a β-Globin PCR as internal control.
A total of 544 clinical samples were used to compare the real-time TaqMan PCR with the traditional Dominguez PCR, the traditional Olerup PCR and a commercial Olerup based HLA-B27 detection kit (Olerup SSPTM HLA-B27, GenoVision). While 542 samples gave concordant results, two samples showed discrepancies and were further analyzed. One sample that showed a discrepancy was negative with the traditional Olerup method and positive with the three other procedures. Sequencing analysis showed the presence of HLA-B2712 in this sample. The other sample, positive with both Olerup based PCRs and negative with both Dominguez based methods, turned out to be positive for HLA-B2707 by sequence analysis.
With a correct result for 543 out of 544 samples (99.8%), we consider our real-time HLA-B27 PCR is a reliable method to detect HLA-B27 in the Dutch population, with reduced hands-on time and contamination risk compared to traditional PCR methods.
人类白细胞抗原 B27（HLA-B27）与强直性脊柱炎密切相关。该疾病患者中有 90%存在 B27 等位基因，而白种人中仅有 9%存在。HLA-B27 的分子检测传统上基于外显子 2（Olerup 方法）或外显子 3（Dominguez 方法）的等位基因特异性扩增，通过 PCR 进行，然后进行凝胶分析。
我们开发了一种基于 Dominguez 方法的实时 TaqMan PCR，带有β-珠蛋白 PCR 作为内对照。
总共使用了 544 个临床样本来比较实时 TaqMan PCR 与传统的 Dominguez PCR、传统的 Olerup PCR 和基于商业的 Olerup 的 HLA-B27 检测试剂盒（Olerup SSPTM HLA-B27，GenoVision）。虽然 542 个样本给出了一致的结果，但有两个样本显示出差异，进一步进行了分析。一个显示差异的样本用传统的 Olerup 方法呈阴性，而用其他三种方法呈阳性。测序分析显示该样本存在 HLA-B2712。另一个样本用两种基于 Olerup 的 PCR 均呈阳性，而用两种基于 Dominguez 的方法均呈阴性，经序列分析结果为 HLA-B2707 阳性。
544 个样本中有 543 个（99.8%）的结果正确，我们认为我们的实时 HLA-B27 PCR 是一种可靠的方法，可以在荷兰人群中检测 HLA-B27，与传统 PCR 方法相比，减少了操作时间和污染风险。
