Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

Abstract

Highly sensitive detection of HIV-1 nucleic acids is of critical importance for evaluating treatment interventions superimposed on combination antiretroviral therapy (cART) in HIV-1 infected individuals. SIV infection of rhesus macaques models many key aspects of human HIV-1 infection and plays a key role in evaluation of approaches for prevention, treatment and attempted eradication of HIV infection. Here we describe ultrasensitive droplet digital PCR (ddPCR) DNA and RT-ddPCR RNA assays for detecting simian immunodeficiency virus (SIV) on the Raindance ddPCR platform. We demonstrate that RainDance ddPCR can tolerate significantly higher cell DNA input without inhibition on a per reaction basis, exceeding the Bio-Rad ddPCR per-reaction input limit by about 18-fold, effectively increasing viral detection sensitivity by allowing a large quantity of sample to be analyzed in each reaction. In addition, the combination of a high processivity RT enzyme and RainDance ddPCR could overcome inhibition in severely inhibited viral RNA samples. These assays offer valuable tools for assessing low level viral production/replication and strategies for targeting residual virus in the setting of cART suppression of viral replication. The methodologies presented here can be adapted for a broad range of applications where highly sensitive nucleic acid detection is required.