Laboratory Branch, Division of HIV/ADS Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
PLoS One. 2007 Jul 25;2(7):e638. doi: 10.1371/journal.pone.0000638.
The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing.
We developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing.
Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations.
众所周知，抗逆转录病毒疗法的成功受到可通过常规批量测序检测到的耐药性 HIV-1 的影响。目前，有必要评估常规基因分型检测下限以下低频耐药变异体的临床后果。然而，耐药亚群的敏感检测需要用于常规检测的简单实用方法。
我们开发了九种关键耐药突变的高灵敏度和简单实时 PCR 检测方法，并表明这些检测方法克服了 HIV-1 临床标本中的大量序列异质性。我们专门使用了抗逆转录病毒药物时代之前的早期野生型病毒样本来测量背景反应性，并能够定义高度特异性的筛选截止值，其灵敏度比常规基因分型高 67 倍。我们还证明，对突变特异性 PCR 产物进行测序提供了一种直接且新颖的策略，可进一步检测和关联相关耐药突变，从而易于识别多药耐药变体。通过克隆测序验证了突变特异性扩增子序列中揭示的耐药突变关联。
综合敏感实时 PCR 检测和突变特异性扩增子测序提供了一种强大而简单的方法，可提高 HIV-1 耐药突变的检测和评估。
