Center for Global Infectious Disease Research, Seattle Children's Research Institute, Seattle, Washington, USA.
Department of Bioengineering, University of Washington, Seattle, Washington, USA.
AIDS Res Hum Retroviruses. 2021 Dec;37(12):930-935. doi: 10.1089/AID.2021.0103. Epub 2021 Nov 30.
The nucleoside reverse transcriptase inhibitor abacavir (ABC) is used commonly to treat young children with HIV infection and is a component of the fixed-dose-combination Triumeq. ABC can trigger a severe hypersensitivity reaction in people who are homozygous or heterozygous for . Testing for before ABC initiation is standard-of-care in high-resource settings, but current tests are costly or difficult to access in resource-limited settings. To address these gaps, we developed an inexpensive simple-to-use rapid assay to detect . We designed and optimized a multiplexed polymerase chain reaction (PCR) to amplify subtypes and the human beta-globin gene; employed probes and ligation to specifically tag the allele with biotin. Tagged-ligated products were detected by immunocapture in an enzyme-linked immunosorbent assay plate or lateral flow strip. Cell lines with known HLA genotypes were used to optimize the assay. The optimized assay was then compared with genotypes of clinical specimens ( = 60) determined by sequencing, with specimens enriched for individuals with . The optimized assay utilizes 40-min 35-cycle multiplex PCR for and beta-globin; 20-min ligation reaction; and 15-min detection. Evaluation of the oligonucleotide ligation assay using clinical specimens had a sensitivity of 100% ( = 27/27 typed as ) and specificity of 100% ( = 33/33 typed as non-) by visual interpretation of lateral flow strips. The cost is US$5.96/specimen. This rapid and economical assay accurately detects in clinical specimens. Use of this assay could expand access to genotyping and facilitate safe same-day initiation of ABC-based treatment.
核苷类逆转录酶抑制剂阿巴卡韦（ABC）常用于治疗感染 HIV 的婴幼儿，也是固定剂量复方制剂 Triumeq 的组成部分。ABC 可在纯合子或杂合子携带 HLA-B57:01 人群中引发严重超敏反应。在资源丰富的环境中，在使用 ABC 之前进行 HLA-B57:01 检测是标准的护理方法，但目前的检测在资源有限的环境中成本较高或难以获得。为了解决这些差距，我们开发了一种廉价、易于使用的快速检测方法来检测 HLA-B57:01。我们设计并优化了一种多重聚合酶链反应（PCR），以扩增 HLA-B57:01 亚型和人类β-球蛋白基因；采用探针和连接技术，用生物素特异性标记 HLA-B57:01 等位基因。标记的连接产物通过酶联免疫吸附试验板或侧向流动条中的免疫捕获进行检测。使用具有已知 HLA 基因型的细胞系来优化该检测方法。然后，将优化的检测方法与通过测序确定的临床标本（ = 60）的基因型进行比较，标本中富集了 HLA-B57:01 阳性个体。优化后的检测方法利用 40 分钟 35 个循环的多重 PCR 进行 HLA-B57:01 和β-球蛋白扩增；20 分钟的连接反应；和 15 分钟的检测。使用临床标本评估寡核苷酸连接检测法，通过对侧向流动条的视觉解释，其灵敏度为 100%（ = 27/27 型为 ），特异性为 100%（ = 33/33 型为非 ）。成本为 5.96 美元/标本。这种快速且经济的检测方法可准确检测临床标本中的 HLA-B57:01。该检测方法的使用可以扩大 HLA-B*57:01 基因分型的可及性，并有助于安全地在当天开始基于 ABC 的治疗。
