Improved Single-Copy Assays for Quantification of Persistent HIV-1 Viremia in Patients on Suppressive Antiretroviral Therapy
Improved Single-Copy Assays for Quantification of Persistent HIV-1 Viremia in Patients on Suppressive Antiretroviral Therapy
复制标题
DOI：
10.1128/jcm.02060-14
复制
发表时间：
2014-11-01
影响因子：
9.4
通讯作者：
Mellors, John W.
中科院分区：
文献类型：
--
作者：
Cillo, Anthony R.;Vagratian, David;Mellors, John W.
针对 HIV-1 gag RNA 的单拷贝敏感性定量实时 PCR (qRT-PCR) 测定（gag 单拷贝测定 [gSCA]）已被广泛用于对接受有效抗逆转录病毒治疗 (ART) 的患者进行低于临床测定检测限的血浆病毒血症定量，但由于引物/探针的原因，15% 至 30% 的样本中的病毒 RNA 扩增效率低下 不匹配。我们试图通过提高核酸回收率、设计 qRT-PCR 引物和针对 HIV-1 pol 基因中高度保守的整合酶区域的探针（整合酶单拷贝测定 [iSCA]）以及增加测试的血浆体积（Mega-iSCA）来开发改进的单拷贝测定，从而提高灵敏度。我们在来自 10 名连续病毒血症> 1,000 拷贝/ml 的患者和 25 名连续接受抑制性 ART 患者的配对血浆样本中评估了 gSCA 与 iSCA。与 Roche Cobas Ampliprep/TaqMan 2.0 相比，10 个病毒血症样本中的 3 个使用 gSCA 扩增效率较低，而所有 10 个样本使用 iSCA 扩增均有效。在接受抑制性 ART 的患者的 25 个样本中，gSCA 检测出 HIV-1 RNA 呈阴性的 12 个样本中的 8 个通过 iSCA 可检测到 HIV-1 RNA，并且在 25 个样本中的 10 个样本中，iSCA 检测到的 HIV-1 RNA 水平是 gSCA 的 3 倍或更高。使用 Mega-iSCA 对 7 名患者的大容量血浆样本（> 20 ml）进行了检测，其中 6 名患者的 HIV-1 RNA 可以定量，其中 5 名患者中的 4 名通过标准体积 iSCA 呈阴性。这些具有卓越灵敏度的改进检测方法将有助于评估体内干预措施是否可以减少血浆病毒血症，以及评估残留病毒血症与其他病毒学参数（包括诱导型原病毒库）之间的关系。
A quantitative real-time PCR (qRT-PCR) assay with single-copy sensitivity targeting HIV-1 gag RNA (the gag single-copy assay [gSCA]) has been used widely to quantify plasma viremia below the limit of detection of clinical assays in patients on effective antiretroviral therapy (ART), but viral RNA in 15 to 30% of samples amplifies inefficiently because of primer/probe mismatches. We sought to develop improved single-copy assays with increased sensitivity by improving nucleic acid recovery, designing qRT-PCR primers and a probe for a highly conserved region of integrase in the HIV-1 pol gene (the integrase single-copy assay [iSCA]), and increasing the plasma volume tested (Mega-iSCA). We evaluated gSCA versus iSCA in paired plasma samples from 10 consecutive patients with viremia of >1,000 copies/ml and 25 consecutive patients on suppressive ART. Three of 10 viremic samples amplified inefficiently with gSCA compared to the Roche Cobas Ampliprep/TaqMan 2.0, whereas all 10 samples amplified efficiently with iSCA. Among 25 samples from patients on suppressive ART, 8 of 12 samples that were negative for HIV-1 RNA by gSCA had detectable HIV-1 RNA by iSCA, and iSCA detected 3-fold or higher HIV-1 RNA levels compared to gSCA in 10 of 25 samples. Large-volume plasma samples (>20 ml) from 7 patients were assayed using Mega-iSCA, and HIV-1 RNA was quantifiable in 6, including 4 of 5 that were negative by standard-volume iSCA. These improved assays with superior sensitivity will be useful for evaluating whether in vivo interventions can reduce plasma viremia and for assessing relationships between residual viremia and other virologic parameters, including the inducible proviral reservoir.