Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR

Abstract

Droplet digital PCR (ddPCR) is an emerging nucleic acid detection method that provides absolute quantitations of target sequences without relying on the use of standard curves. The ability of ddPCR to detect and quantitate total HIV-1 DNA and 2-LTR circles from a panel of patients on and off antiviral therapy was evaluated compared to established real-time (RT)-PCR methods. To calculate the dynamic range of ddPCR for HIV-1 DNA and 2-LTR circles, serial dilutions of DNA amplicons or episomes were determined by ddPCR as well as with RT-PCR. HIV-1 DNA from 3 viremic patients and 4 patients on suppressive antiretroviral therapy, and 2-LTR circles from 3 patients with low-level viremia were also quantitated. Copy numbers determined by ddPCR of serial dilutions of HIV-1 or human CCR5 DNA amplicon standards were comparable to nominal input copy number. The sensitivity of ddPCR to detect HIV-1 or CCR5 DNA was similar to that of RT-PCR. Low levels of 2-LTR circles were detected in samples from all 3 patients by both ddPCR and RT-PCR. ddPCR is a promising novel technology for the study of HIV-1 reservoirs and persistence, but further optimization of this novel technology would enhance the detection of very low-level viral genetic targets.

Figure 1

Visual representation of ddPCR results for HIV-1 DNA standards after flow enumeration. Positive droplets (upper cluster) and negative droplets (lower cluster) are shown for 6 serially diluted linear DNA standards (A1 through F1) and negative control (G1). The Y axis represents fluorescent intensity and the X axis is the total number of events for each standard dilution. The manually set threshold for droplet positivity is represented by the horizontal line.