Clin Lab. 2022 Nov 1;68(11). doi: 10.7754/Clin.Lab.2022.220231.
The human leukocyte antigen (HLA) class I gene, the B locus, allele 27, HLA-B27 is one of the most fascinating risk factors that is strongly associated with developing spondyloarthropathies (SpA). HLA-B27 testing has been routinely available in the diagnosis of those diseases. This study aimed to develop a fluorogenic real-time PCR and to compare it with PCR-SSP to detect the HLA-B27 allele among Thai blood donors.
A total of 391 DNA samples were obtained from Thai blood donors at Thammasat University Hospital and tested for HLA-B27 allele detection. A new real-time PCR was developed and validated to identify this allele and subsequently compared with those results tested with PCR-SSP. The sensitivity of detection was performed using known HLA-B27-positive and -negative samples with concentrations ranging from 0.001 to 100 ng/µL. Additionally, HLA-B27 subtyping was performed by DNA sequencing containing second and third exons of this gene among all the HLA-B*27-positive donors.
The validity of real-time PCR using known DNA controls and the results obtained by PCR-SSP techniques were in 100% concordance. The method was sensitive even at low DNA concentrations (1 ng/µL). Of 391 donors, 24 (6.14%; 95% CI, 3.97 - 9.00) were found to have the HLA-B27 allele, while the remaining 367 (93.86%; 95% CI, 91.00 - 96.03) did not have this allele. Donors presented HLA-B27-positive, HLA-B27:06, the most common allele, followed by HLA-B27:04, -B27:05, and -B27:07.
HLA-B*27 using fluorogenic real-time qualitative PCR was found to be superior compared with that of PCR-SSP. The method is rapid, accurate, reliable, and sensitive for detection. In addition, this method provides convenience in the early treatment of SpA patients and relieves their suffering.
人类白细胞抗原（HLA）I 类基因 B 座，等位基因 27，HLA-B27 是与脊柱关节病（SpA）发展强烈相关的最引人注目的危险因素之一。HLA-B27 检测已常规用于这些疾病的诊断。本研究旨在开发一种荧光实时 PCR 并将其与 PCR-SSP 进行比较，以检测泰国献血者中的 HLA-B27 等位基因。
从 Thammasat 大学医院的泰国献血者中获得了 391 个 DNA 样本，并对其 HLA-B27 等位基因检测进行了测试。开发了一种新的实时 PCR 并对其进行了验证，以鉴定该等位基因，并随后与使用 PCR-SSP 测试的结果进行了比较。使用浓度范围为 0.001 至 100 ng/µL 的已知 HLA-B27 阳性和阴性样本进行了检测灵敏度。此外，对所有 HLA-B*27 阳性供体的该基因第二和第三外显子进行了 HLA-B27 亚型分析。
使用已知 DNA 对照和 PCR-SSP 技术获得的实时 PCR 有效性完全一致。即使在低 DNA 浓度（1 ng/µL）时，该方法也具有很高的灵敏度。在 391 名供体中，发现 24 名（6.14%；95%置信区间，3.97-9.00）具有 HLA-B27 等位基因，而其余 367 名（93.86%；95%置信区间，91.00-96.03）没有该等位基因。供体表现出 HLA-B27 阳性，HLA-B27：06 是最常见的等位基因，其次是 HLA-B27：04、-B27：05 和 -B27：07。
使用荧光实时定性 PCR 的 HLA-B*27 被发现优于 PCR-SSP。该方法快速、准确、可靠且灵敏。此外，该方法为 SpA 患者的早期治疗提供了便利，并减轻了他们的痛苦。
