用于 HIV-1 耐药性检测的内部多重 PCR 分析法的性能--一种更便宜的替代方法。

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods Pub Date : 2024-09-18 DOI:10.1016/j.jviromet.2024.115034

Tumelo L. Fortuin , Paballo Nkone , Allison Glass , Raquel Viana , Keitumetse Moeng , Shayne Loubser , Caroline T. Tiemessen , Simnikiwe H. Mayaphi

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引用次数: 0

摘要

背景：目前，大多数 HIV 耐药性 PCR 检测方法都是将蛋白酶逆转录酶（PR-RT）片段与整合酶（IN）片段分开扩增。本研究旨在开发一种同时扩增 PR-RT 和 IN 片段的多重 PCR 检测方法，用于 HIV-1 耐药性检测：方法：对从国家卫生实验室服务处（NHLS）和 Lancet 实验室提取的总核酸进行了内部多重 PCR 检测评估。对扩增子进行桑格测序，并使用 HIV 斯坦福耐药性数据库评估 HIV-1 耐药性突变（DRMs）：本研究检测了 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 实验室，18 份来自 NHLS 实验室。内部多重 PCR 检测法在大多数样本中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%）。在 IN DRMs 方面，Lancet 检测与内部检测序列数据的一致性为 100%，但与 PR-RT 的一致性较低（87.0%）。内部多重 PCR 检测的精确度和可重复性分析表明，PR-RT 和 IN 片段的序列相似度≥99.9%，DRM 结果相似：内部多重 PCR 检测法在 IN 片段扩增方面表现出令人满意的性能和更高的灵敏度。这可能是一种经济有效的 HIV-1 耐药性检测方法，因为在大多数样本中，PR-RT 和 IN 片段都能在一个反应中成功扩增。

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Performance of an in-house multiplex PCR assay for HIV-1 drug resistance testing – A cheaper alternative

Background

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.

Methods

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.

Results

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.

Conclusions

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.

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来源期刊

Journal of virological methods 医学-病毒学

CiteScore

5.80

自引率

0.00%

发文量

209

审稿时长

41 days

期刊介绍： The Journal of Virological Methods focuses on original, high quality research papers that describe novel and comprehensively tested methods which enhance human, animal, plant, bacterial or environmental virology and prions research and discovery. The methods may include, but not limited to, the study of: Viral components and morphology- Virus isolation, propagation and development of viral vectors- Viral pathogenesis, oncogenesis, vaccines and antivirals- Virus replication, host-pathogen interactions and responses- Virus transmission, prevention, control and treatment- Viral metagenomics and virome- Virus ecology, adaption and evolution- Applied virology such as nanotechnology- Viral diagnosis with novelty and comprehensive evaluation. We seek articles, systematic reviews, meta-analyses and laboratory protocols that include comprehensive technical details with statistical confirmations that provide validations against current best practice, international standards or quality assurance programs and which advance knowledge in virology leading to improved medical, veterinary or agricultural practices and management.