Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea.
Clin Chim Acta. 2011 Oct 9;412(21-22):1912-7. doi: 10.1016/j.cca.2011.06.034. Epub 2011 Jul 7.
Real-time PCR and melting curve analysis is the relatively recent method for HLA-B27 genotyping, which has advantages of being simple and rapid.
The accuracy of melting curve analysis for HLA-B27 was assessed in 153 clinical samples and 52 DNA samples from International Histocompatibility Workshop (IHW) cell lines, with sequence-based typing (SBT) as the reference method. We predicted melting reaction for various HLA-B27 subtypes using simulation software.
For clinical samples, 53 HLA-B27-positive and 100 negative results by melting curve analysis were confirmed by completely concordant SBT results. The B27:05 allele was found in 50 patients, and the B27:04 allele in 3 patients. Among 62 known alleles, 21 alleles had differences in the target sequence, including 10 alleles having mismatches in the primer binding site. In these alleles, differences in melting points (T(m)) were predicted to be ≤ 1.2°C. The predicted results were obtained when IHW samples were tested, which revealed slight lower T(m) for B27:06 and negative results for B27:07.
Genotyping of HLA-B27 by melting curve analysis was fast and reliable for routine laboratory testing for frequent alleles. In silico melting simulations provided useful information about the utility and limitation of this method for diverse HLA-B27 alleles.
实时 PCR 和熔解曲线分析是 HLA-B27 基因分型的相对较新方法，具有简单快速的优点。
使用基于序列的分型 (SBT) 作为参考方法，评估熔解曲线分析对 153 个临床样本和 52 个国际组织相容性工作坊 (IHW) 细胞系 DNA 样本中 HLA-B27 的准确性。我们使用模拟软件预测了各种 HLA-B27 亚型的熔解反应。
对于临床样本，通过熔解曲线分析得到的 53 个 HLA-B27 阳性和 100 个阴性结果与完全一致的 SBT 结果相吻合。在 50 名患者中发现了 B27:05 等位基因，在 3 名患者中发现了 B27:04 等位基因。在已知的 62 个等位基因中，有 21 个在目标序列上存在差异，包括 10 个在引物结合位点存在错配。在这些等位基因中，预测熔点 (T(m)) 的差异≤1.2°C。对 IHW 样本进行测试时得到了预测结果，结果显示 B27:06 的 T(m) 略低，B27:07 则为阴性。
熔解曲线分析对 HLA-B27 的基因分型对于常规实验室检测常见等位基因快速可靠。计算机模拟熔解实验提供了有关该方法在不同 HLA-B27 等位基因中的应用和局限性的有用信息。
