Adaptation of Droplet Digital PCR-Based HIV Transcription Profiling to Digital PCR and Association of HIV Transcription and Total or Intact HIV DNA.
Adaptation of Droplet Digital PCR-Based HIV Transcription Profiling to Digital PCR and Association of HIV Transcription and Total or Intact HIV DNA.
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DOI：
10.3390/v15071606
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发表时间：
2023-07-22
期刊：
影响因子：
--
通讯作者：
Telwatte S
中科院分区：
文献类型：
--
作者：
Tumpach C;Rhodes A;Kim Y;Ong J;Liu H;Chibo D;Druce J;Williamson D;Hoh R;Deeks SG;Yukl SA;Roche M;Lewin SR;Telwatte S
在大多数接受有效抗逆转录病毒治疗（ART）的艾滋病毒感染者（PLWH）中，尽管没有病毒血症，但在CD 4 + T细胞中很容易检测到细胞相关病毒转录本。HIV RNA种类的定量提供了对ART期间在整个身体的细胞和组织中持续存在的前病毒（“HIV病毒”）的转录活性的了解。Yukl实验室开发的一种用于HIV RNA定量的技术“HIV转录谱”使用液滴数字PCR测量一系列HIV RNA种类。为了利用数字（d）PCR的进步，我们将“HIV转录谱”技术应用于Qiagen的dPCR平台（QIAcuity），并将其性能与液滴数字（dd）PCR（Bio-Rad QX 200系统）进行比较。使用RNA标准品，平行测试了这两种技术，并评估了多个参数，包括灵敏度、特异性、线性以及试验内和试验间变异性。然后将新验证的dPCR检测应用于PLWH样本，以确定相对于HIV储库大小的HIV转录活性。我们报告说，HIV转录谱很容易适应dPCR和检测进行类似的ddPCR，没有差异的检测特征。我们将这些检测应用于23例PLWH的队列，发现基于遗传完整的前病毒DNA的HIV储库大小不能预测HIV转录活性。相比之下，总DNA水平与大多数HIV转录物（起始、近端和远端伸长、未剪接和完成，但不是多重剪接）的水平相关，这表明相当大比例的HIV转录物可能来源于有缺陷的前病毒。这些发现可能对测量和评估治疗策略和临床试验结果具有影响。
In most people living with HIV (PLWH) on effective antiretroviral therapy (ART), cell-associated viral transcripts are readily detectable in CD4+ T cells despite the absence of viremia. Quantification of HIV RNA species provides insights into the transcriptional activity of proviruses that persist in cells and tissues throughout the body during ART (‘HIV reservoir’). One such technique for HIV RNA quantitation, ‘HIV transcription profiling’, developed in the Yukl laboratory, measures a series of HIV RNA species using droplet digital PCR. To take advantage of advances in digital (d)PCR, we adapted the ‘HIV transcription profiling’ technique to Qiagen’s dPCR platform (QIAcuity) and compared its performance to droplet digital (dd)PCR (Bio-Rad QX200 system). Using RNA standards, the two technologies were tested in parallel and assessed for multiple parameters including sensitivity, specificity, linearity, and intra- and inter-assay variability. The newly validated dPCR assays were then applied to samples from PLWH to determine HIV transcriptional activity relative to HIV reservoir size. We report that HIV transcriptional profiling was readily adapted to dPCR and assays performed similarly to ddPCR, with no differences in assay characteristics. We applied these assays in a cohort of 23 PLWH and found that HIV reservoir size, based on genetically intact proviral DNA, does not predict HIV transcriptional activity. In contrast, levels of total DNA correlated with levels of most HIV transcripts (initiated, proximally and distally elongated, unspliced, and completed, but not multiply spliced), suggesting that a considerable proportion of HIV transcripts likely originate from defective proviruses. These findings may have implications for measuring and assessing curative strategies and clinical trial outcomes.
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影响因子：
16.6
作者：
Kinloch NN;Ren Y;Conce Alberto WD;Dong W;Khadka P;Huang SH;Mota TM;Wilson A;Shahid A;Kirkby D;Harris M;Kovacs C;Benko E;Ostrowski MA;Del Rio Estrada PM;Wimpelberg A;Cannon C;Hardy WD;MacLaren L;Goldstein H;Brumme CJ;Lee GQ;Lynch RM;Brumme ZL;Jones RB
通讯作者：
Jones RB
影响因子：
30.3
作者：
Li, Shuying S.;Hickey, Andrew;Shangguan, Shida;Ehrenberg, Philip K.;Geretz, Aviva;Butler, Lauryn;Kundu, Gautam;Apps, Richard;Creegan, Matthew;Clifford, Robert J.;Pinyakorn, Suteeraporn;Eller, Leigh Anne;Luechai, Pikunchai;Gilbert, Peter B.;Holtz, Timothy H.;Chitwarakorn, Anupong;Sacdalan, Carlo;Kroon, Eugene;Phanuphak, Nittaya;de Souza, Mark;Ananworanich, Jintanat;O'Connell, Robert J.;Robb, Merlin L.;Michael, Nelson L.;Vasan, Sandhya;Thomas, Rasmi
通讯作者：
Thomas, Rasmi
影响因子：
158.5
作者：
Gao, XJ;Nelson, GW;Carrington, M
通讯作者：
Carrington, M
影响因子：
15.9
作者：
Lee, Guinevere Q.;Orlova-Fink, Nina;Lichterfeld, Mathias
通讯作者：
Lichterfeld, Mathias
影响因子：
6.4
作者：
KLEIN, MR;KEET, IPM;MIEDEMA, F
通讯作者：
MIEDEMA, F