Abstract
Accurate and sensitive quantification of rebound competent HIV that persists despite combination antiretroviral treatment (cART), including in latently infected cells (i.e., viral reservoir), is critical for evaluating cure strategies for decreasing or eliminating this reservoir. Simian immunodeficiency virus (SIV)-infected Rhesus macaques are an important non-human primate (NHP) system for studying potential cure strategies as they model many key aspects of human HIV-infection including the persistence of a latent viral reservoir in resting memory CD4+ T cells in animals receiving prolonged cART. In this report, we describe the design and testing of a sensitive SIV droplet digital PCR (ddPCR) assay through exploring the combination and optimization of different probe systems (including single, double quencher probes and minor groove binder (MGB) probes) and reaction conditions to eliminate background signal(s), ensure distinct target signal cluster separation from non-target signals, and enable detection and quantification of low level authentic target signals. Similar reaction conditions and assay validation procedures can be explored for potential development of additional assays for other applications that require sensitive detection of low-level targets in a large background of nucleic acid input derived from cell or tissue sources.
Highlights
Droplet digital PCR is a nucleic acid detection method that provides absolute quantification of specific targets by partitioning a standard quantitative PCR reaction into tens of thousands to millions of individual droplets of nanoliter or picoliter size
The advantage of this sample type was that the Simian immunodeficiency virus (SIV) signal was preamplified, and its corresponding Droplet digital PCR (ddPCR) signal clusters were easy to detect and compare among different test conditions due to the large signal counts
These samples were identical in genetic background complexity to test samples that are usually encountered in non-human primate (NHP) research
Summary
Droplet digital PCR (ddPCR) is a nucleic acid detection method that provides absolute quantification of specific targets by partitioning a standard quantitative PCR reaction into tens of thousands to millions of individual droplets of nanoliter or picoliter size. The system is designed to operate such that each droplet contains a single target molecule or no target. One of the main advantages of ddPCR platforms compared to qPCR platforms is the capability for absolute quantification without the need for a standard curve. This feature allows effective comparison among quantitative measurements and quality control of routine
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