Quantification of HIV DNA Using Droplet Digital PCR Techniques

Abstract

HIV persists, despite effective antiretroviral therapy, in long-lived cells, posing a major barrier toward 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. 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 and then 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. © 2018 by John Wiley & Sons, Inc.

Keywords: HIV DNA quantification • human immunodeficiency virus (HIV) • viral DNA integration.

Figure 1

ddPCR workflow: First a PCR mix containing target DNA along with primers and fluorescently labeled probes is partitioned into around 20,000 nanoliter sized droplets with the QX200 droplet generator. Then these droplets are cycled to an endpoint on a thermal cycler. Next the droplets are read with the QX200 Droplet Reader which sips each well, singulates the droplets and passes them by a two-color detection system. The data generated can finally be analyzed in the quantasoft software where thresholds should be set between the negative and positive droplets.

Figure 2

ddPCR Plate Layout: A typical layout for droplet generation and amplification. Samples should be run in triplicate. DG8 cartridge can accommodate 8 samples at a time so it is convenient to organize samples by columns. All runs should include positive and negative controls, which could be DNA extracted from HIV negative cells such as the cell line CEM and DNA extracted from HIV positive cells such as the ACH2 cell line.

Figure 3

Schematic of ddPCR primer/probe target regions in the HIV genome for HIV DNA quantification.

Figure 4

Schematic of DG8 cartridge for droplet generation: The droplet cartridges are composed of microfluidic channels. Sample is loaded into the center chamber of the cartridge, and oil is loaded into the bottom chamber then a gasket is applied and the cartridge plus holder are transferred to the droplet generator. The droplet generator applies vacuum pressure to pull samples and oil though these microfluidic channels. The samples and oil meet at a T-junction where they form water-in-oil droplets which then collect in the last chamber.

Figure 5

Dynamic Range of ddPCR platform: A.) ddPCR accurately quantifies targets from 0.3 to 1x10⁵ copies (mean 2.2-fold variation from expected) but was inaccurate above 1x10⁵ copies due to saturation of positive droplets. RNA transcripts were serially diluted, reversed transcribed, and quantified using ddPCR. Measured copies/well are compared to the expected line. B.) RNA transcripts were serially diluted from 100 to 0.3cps/well, reversed transcribed, and quantified using ddPCR. The number next to each point shows the average of the 10 replicates; the error bar is ±1 standard deviation (SD). Note that the error bar is very close to that predicted for a Poisson distribution, an irreducible minimum.

Figure 6

Trouble shooting example of sheared droplets: HIV RNA transcript standards were serially diluted and droplets were generate for each dilution. Droplets in the 8 wells to the left were sheared due abrupt and fast pipetting. Sheared droplets are easily distinguished with ‘rain’ under the negative droplet populations (bracket). Rain under the positive droplets is typically seen and may be attributed to inefficient PCR amplification. The 8 wells on the right show properly handled droplets having minimal rain under both the positive and negative droplets.

Figure 7

Setting the threshold: HIV RNA transcript standards were serially diluted and ddPCR was performed for each dilution. The data generated was then analyzed by setting varying thresholds. 10 increments were determined to set threshold bars ranging from the mean of fluorescence intensity of the negative droplets (mean: 9800) to under the mean fluorescence intensity of the positive droplets (mean: 17832). Increments also included a value that was 50% above the negative fluoresce (14700) and a value that would capture 98% of all positives (15600). Once above the negative droplets the overall HIV concentration measured with ddPCR does not significantly change even when adjusting the threshold level.

Figure 8

Trouble shooting example variance in mean fluorescence due to primer/probe mismatches: Primer/probe mismatches lead to a decrease in the mean fluorescence amplitude of positive droplets. A. Quantification schematic from a ddPCR run on patient plasma from three individuals compared to an RNA transcript control. B. The number of polymorphisms in the probe target region obtained by population sequencing and the corresponding mean amplitude of fluorescence in positive droplets obtained by ddPCR. C. Highlighter plot showing the population based and single genome sequences obtained from patient derived plasma from three individuals with the PCR region of interest compared to the primer sequences.