Department of Medical Virology, University of Pretoria, South Africa.
Lancet laboratories, South Africa.
J Virol Methods. 2024 Dec;330:115034. doi: 10.1016/j.jviromet.2024.115034. Epub 2024 Sep 18.
Currently, most HIV drug resistance PCR assays amplify the protease-reverse transcriptase (PR-RT) fragment separately from the integrase (IN) fragment. The aim of this study was to develop a multiplex PCR assay that simultaneously amplifies PR-RT and IN fragments for HIV-1 drug-resistance testing.
The in-house multiplex PCR assay was evaluated on extracted total nucleic acids obtained from the National Health Laboratory Service (NHLS) and Lancet laboratories. Sanger sequencing was performed on amplicons, and HIV-1 drug-resistance mutations (DRMs) were assessed using HIV Stanford drug resistance database.
This study tested 59 patient samples with known HIV-1 viral load and DRM results; 41 from Lancet and 18 from NHLS. In-house multiplex PCR assay detected one or both fragments in most samples but had higher sensitivity for detection of IN fragment (93.2 %) compared to PR-RT fragment (83.1 %). There was 100 % concordance between Lancet assay versus in-house assay sequence data for IN DRMs, but lower concordance with PR-RT (87.0 %). The in-house multiplex PCR assay's precision and reproducibility analysis showed ≥99.9 % sequence similarity and yielded similar DRM results for both PR-RT and IN fragments.
The in-house multiplex PCR assay demonstrated satisfactory performance and higher sensitivity for IN fragment amplification. This could be a cost-effective method for HIV-1 drug resistance testing as both PR-RT and IN fragments are successfully amplified in one reaction in most samples.
目前，大多数 HIV 耐药性 PCR 检测方法分别从蛋白酶-逆转录酶（PR-RT）片段和整合酶（IN）片段扩增 PR-RT 和 IN 片段。本研究旨在开发一种同时扩增 PR-RT 和 IN 片段用于 HIV-1 耐药性检测的多重 PCR 检测方法。
该实验室内的多重 PCR 检测方法在从国家卫生实验室服务（NHLS）和 Lancet 实验室获得的提取总核酸上进行了评估。对扩增子进行 Sanger 测序，使用 HIV 斯坦福耐药性数据库评估 HIV-1 耐药突变（DRMs）。
本研究共检测了 59 份已知 HIV-1 病毒载量和 DRM 结果的患者样本；其中 41 份来自 Lancet，18 份来自 NHLS。实验室内的多重 PCR 检测方法在大多数样本中均能检测到一个或两个片段，但 IN 片段的检测灵敏度（93.2%）高于 PR-RT 片段（83.1%）。IN DRMs 方面，Lancet 检测方法与实验室内检测方法的序列数据具有 100%的一致性，但与 PR-RT 相比，一致性较低（87.0%）。实验室内多重 PCR 检测方法的精密度和重现性分析显示，序列相似度≥99.9%，且 PR-RT 和 IN 片段的 DRM 结果相似。
实验室内的多重 PCR 检测方法表现出令人满意的性能，对 IN 片段的扩增具有更高的灵敏度。由于在大多数样本中，一个反应即可成功扩增 PR-RT 和 IN 片段，因此该方法可能是一种具有成本效益的 HIV-1 耐药性检测方法。
