Development and optimization of a simian immunodeficiency virus (SIV) droplet digital PCR (ddPCR) assay

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.

SARS-CoV-2 Subgenomic RNAs: Characterization, Utility, and Perspectives

“…Limited yields of cells or fluids from sampling procedures such as nasopharyngeal swabs, and the presence of potential inhibitors (e.g., chemical or protein contaminants) in clinical samples, require that PCR amplification and detection be highly sensitive and reliable during SARS-CoV-2 nucleic acid analysis. Digital PCR has demonstrated significant advantages in both SARS-CoV-2 gRNA [94][95][96] and sgRNA [49,97,98] studies due to its ability for absolute quantification [98][99][100][101][102][103][104][105], tolerance to inhibitors [106], increased precision at low analyte copy numbers [107][108][109], and inter-run reproducibility [110][111][112]. One additional distinct advantage of the digital PCR approach is its lower susceptibility to sequence mismatches, which is especially relevant as emerging mutations that can potentially predominate could affect the performance of real-time PCR-based assays if they occur in regions where the PCR primer and probes are located [113,114].…”

Clinical Application of Droplet Digital PCR for Hepatitis Delta Virus Quantification

“…A high target concentration leads to a saturation of positive droplets, making the Poisson statistic invalid and requiring, for samples with elevated viral load, the addition of an initial dilution step. Indeed, the BioRad ddPCR platform's manufacturer recommends 1 µg of DNA per reaction as the sample input upper limit, while other platforms, such as the Rain-Dance ddPCR system, are able to analyze a larger amount of input DNA per reaction [34]. However, we estimated that the accuracy of recovery was 101%, thus suggesting an acceptable analytical error, and moreover negligible from a clinical point of view.…”

Identifying SARS-CoV-2 Variants of Concern through Saliva-Based RT-qPCR by Targeting Recurrent Mutation Sites

“…We did observe low-level amplification from off-target binding in the E484K, K417T, and L452R assays. High concentrations of minor groove binding probes can produce background fluorescent signal ( 38 ). We could not further investigate if the probe concentration ratio was causing background signal because the commercial kits were premixed.…”