Quantification of HIV DNA Using Droplet Digital PCR Techniques

Abstract

HIV persists despite effective antiretroviral therapy in long lived cells, posing a major barrier towards a cure. A key step in the HIV replication cycle and a hallmark of the Retroviridae family is the integration of the viral DNA into the host genome. Once integrated, HIV expression is regulated by host machinery and the provirus persists until the cell dies. A reservoir of cells harboring replication competent proviruses can survive for years, and mechanisms that maintain that reservoir are under investigation. The majority of integrated proviruses, however, are defective or have large deletions, and the composition of the proviral landscape during therapy remains unknown. Methods to quantify HIV proviruses are useful in investigating HIV persistence, and presented in this unit is a method for total HIV DNA quantification of various HIV genome targets that utilizes the next generation PCR platform, digital PCR. The abundance of various HIV gene targets reflects the overall proviral composition. In this protocol, total genomic DNA is isolated from patient derived cells, which is used as a template for droplet digital PCR in which the PCR reaction is partitioned into approximately 20,000 individual droplets, PCR amplified to an end point, and subjected to absolute quantification by counting the number of positive and negative droplets. Copy number is directly calculated using straightforward Poisson correction. Additionally, this methodological approach can be used to obtain absolute quantification of other DNA targets.

HIV-1 in lymph nodes is maintained by cellular proliferation during antiretroviral therapy

“…HIV-1 quantification. HIV-1 DNA levels were determined using the integrase cell-associated DNA assay (31) or using a droplet digital PCR (ddPCR) assay (42). For ddPCR, HIV-1 DNA copies per million cells were measured in triplicate with a duplexed ddPCR assay measuring the amount of DNA corresponding to HIV-1 gag (43), pol, and the RU5 region of the HIV-1 LTR.…”

Dynamic Shifts in the HIV Proviral Landscape During Long Term Combination Antiretroviral Therapy: Implications for Persistence and Control of HIV Infections

“…DNA extracted from PBMC was quantified using ddPCR assays targeting HIV-1 gag, LTR, and tat/rev regions, and a host gene (CCR5) as previously described [52] (Table S4). To this end, total genomic DNA pellets stored in ethanol were spun at 21,000× g for 10 min and supernatant was removed.…”

“…This assay was multiplexed with previously published oligos in p24 of gag [59] modifying the gag 32t probe to contain an internal ZEN quencher and a 3 Iowa Black FQ (HIV SCA probe 32t ZEN). The first exon of tat was quantified using the forward primer TatRev1_F, reverse primer TatRev1_R, and a Hex tagged TatRev1_probe [52]. The second exon of tat and rev was measured with a forward primer in HIV env called TatRev2_F, a reverse primer in exon 2 of tat/rev: msRNA-R, and a FAM tagged probe in exon 2 of tat/rev: msRNA Probe [52].…”

“…The first exon of tat was quantified using the forward primer TatRev1_F, reverse primer TatRev1_R, and a Hex tagged TatRev1_probe [52]. The second exon of tat and rev was measured with a forward primer in HIV env called TatRev2_F, a reverse primer in exon 2 of tat/rev: msRNA-R, and a FAM tagged probe in exon 2 of tat/rev: msRNA Probe [52]. All ddPCR primer sequences are listed in Table S4, oligos were ordered from Integrated DNA Technologies.…”

“…There are published reports that measure viral DNA load by estimating the numbers of intact proviruses [3,28] and measuring levels of proviruses that are likely to be infectious [45]; in addition, there are methods that quantify either internal portions of the HIV-1 genome or the HIV-1 long terminal repeats (LTRs), but not both simultaneously [23,[46][47][48][49][50][51]. We have developed Droplet Digital PCR (ddPCR) approaches that make it possible to simultaneously quantify both HIV-1 LTRs and internal HIV-1 DNA sequences; these methods are sensitive enough to measure small (2-to 3-fold) differences in relative abundance [52]. We also developed specific primer:probe sets to measure the levels of individual abundant infected clones using primers that flank the site of integration and the provirus.…”