A rapid method for simultaneous detection of phenotypic resistance to inhibitors of protease and reverse transcriptase in recombinant human immunodeficiency virus type 1 isolates from patients treated with antiretroviral drugs
A rapid method for simultaneous detection of phenotypic resistance to inhibitors of protease and reverse transcriptase in recombinant human immunodeficiency virus type 1 isolates from patients treated with antiretroviral drugs
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DOI：
10.1093/jac/42.2.269
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发表时间：
1998-02-01
影响因子：
4.9
通讯作者：
Pauwels, R
中科院分区：
文献类型：
--
作者：
Hertogs, K;de Béthune, MP;Pauwels, R
蛋白酶（PR）和逆转录酶（RT）抑制剂联合治疗可以有效抑制人类免疫缺陷病毒（HIV）的复制，但耐药变异的出现与治疗失败密切相关。在这里，我们描述了一种高通量分析临床样本的新方法，该方法可以通过重组病毒测定技术同时检测HN 1型（HIV-1）对RT和PR抑制剂的表型抗性。从血浆样本中提取HIV-1 RNA，并通过嵌套逆转录pcr扩增出包含整个HIV-1 pr和rt编码序列的2.2 kb片段。然后将PR-RT编码序列库与pGEMT3 Delta PRT质粒共转染到CD4+ T淋巴细胞（MT4）中，其中大部分PR（密码子10 ~ 99）和RT（密码子1 ~ 482）序列被删除。同源重组导致产生嵌合病毒，其中包含来自血浆中HIV-1 RNA的PR和rt编码序列。嵌合病毒对所有目前可用的RT和/或PR抑制剂的敏感性是通过基于MT4细胞-3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑的细胞活力测定在自动化系统中确定的，该系统允许高样品通量。对所有RT和PR抑制剂的耐药性在单个PR-RT抗病毒图中以图形方式显示。该检测系统便于在一次标准化检测中快速大规模测定所有RT和PR抑制剂的表型抗性。
Combination therapy with protease (PR) and reverse transcriptase (RT) inhibitors can efficiently suppress human immunodeficiency virus (HIV) replication, but the emergence of drug-resistant variants correlates strongly with therapeutic failure. Here we describe a new method for high-throughput analysis of clinical samples that permits the simultaneous detection of HN type 1 (HIV-1) phenotypic resistance to both RT and PR inhibitors by means of recombinant virus assay technology. HIV-1 RNA is extracted from plasma samples, and a 2.2-kb fragment containing the entire HIV-1 PR-and RT-coding sequence is amplified by nested reverse transcription-PCR. The pool of PR-RT-coding sequences is then cotransfected into CD4+ T lymphocytes (MT4) with the pGEMT3 Delta PRT plasmid from which most of the PR (codons 10 to 99) and RT (codons 1 to 482) sequences are deleted. Homologous recombination leads to the generation of chimeric viruses containing PR- and RT-coding sequences derived from HIV-1 RNA in plasma. The susceptibilities of the chimeric viruses to all currently available RT and/or PR inhibitors is determined by an MT4 cell-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based cell viability assay in an automated system that allows high sample throughput. The profile of resistance to all RT and PR inhibitors is displayed graphically in a single PR-RT-Antivirogram. This assay system facilitates the rapid large-scale phenotypic resistance determinations for all RT and PR inhibitors in one standardized assay.