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HIV reservoir quantification by five-target multiplex droplet digital PCR.

ABSTRACT: Most latent human immunodeficiency virus (HIV) proviruses are defective and cannot produce infectious virions. Thus, the number of HIV proviruses with intact genomes is a relevant clinical parameter to assess therapies for HIV cure. We describe high-molecular-weight DNA isolation, followed by restriction enzyme fragmentation that limits cutting within the HIV genome. Multiplexed droplet digital PCR quantifies five targets spanning the HIV genome to estimate potentially intact proviral copies. A reference assay counts the number of T lymphocytes and assesses the level of DNA shearing. For complete details on the use and execution of this protocol, please refer to Levy et al. (2021).

SUBMITTER: Levy CN

PROVIDER: S-EPMC8517383 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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HIV reservoir quantification by five-target multiplex droplet digital PCR.

Levy Claire N CN Hughes Sean M SM Roychoudhury Pavitra P Amstuz Chelsea C Zhu Haiying H Huang Meei-Li ML Lehman Dara A DA Jerome Keith R KR Hladik Florian F

STAR protocols 20211011 4

Most latent human immunodeficiency virus (HIV) proviruses are defective and cannot produce infectious virions. Thus, the number of HIV proviruses with intact genomes is a relevant clinical parameter to assess therapies for HIV cure. We describe high-molecular-weight DNA isolation, followed by restriction enzyme fragmentation that limits cutting within the HIV genome. Multiplexed droplet digital PCR quantifies five targets spanning the HIV genome to estimate potentially intact proviral copies. A ...[more]

PMID: 34693363

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Project description:Cell-associated (CA) HIV-1 RNA is considered a potential marker for assessment of viral reservoir dynamics and antiretroviral therapy (ART) response in HIV-infected patients. Recent studies employed sensitive seminested real-time quantitative (q)PCR to quantify CA HIV-1 RNA. Digital PCR has been recently described as an alternative PCR-based technique for absolute quantification with higher accuracy compared to qPCR. Here, a comparison was made between the droplet digital PCR (ddPCR) and the seminested qPCR for quantification of unspliced (us) and multiply spliced (ms) CA HIV-1 RNA. Synthetic RNA standards and CA HIV-1 RNA from infected patients on and off ART (N = 34) were quantified with both methods. Correlations were observed between the methods both for serially diluted synthetic standards (usRNA: R2 = 0.97, msRNA: R2 = 0.92) and patient-derived samples (usRNA: R2 = 0.51, msRNA: R2 = 0.87). Seminested qPCR showed better quantitative linearity, accuracy and sensitivity in the quantification of synthetic standards than ddPCR, especially in the lower quantification ranges. Both methods demonstrated equally high detection rate of usRNA in patient samples on and off ART (91%), whereas ddPCR detected msRNA in larger proportion of samples from ART-treated patients (p = 0.13). We observed an average agreement between the methods for usRNA quantification in patient samples, albeit with a large standard deviation (bias = 0.05±0.75 log10). However, a bias of 0.94±0.36 log10 was observed for msRNA. No-template controls were consistently negative in the seminested qPCR, but yielded a positive ddPCR signal for some wells. Therefore, the false positive signals may have affected the detection power of ddPCR in this study. Digital PCR is promising for HIV nucleic acid quantification, but the false positive signals need further attention. Quantitative assays for CA HIV RNA have the potential to improve monitoring of patients on ART and to be used in clinical studies aimed at HIV eradication, but should be cross-validated by multiple laboratories prior to wider use.

Dataset Information

HIV reservoir quantification by five-target multiplex droplet digital PCR.

Publications

HIV reservoir quantification by five-target multiplex droplet digital PCR.

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