Droplet digital PCR for the precise quantification of HTLV-1

Abstract

Human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that is the etiologic agent in a clinical neuromyelopathy known as HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Quantification of the amount of virus in patient samples is critical for monitoring infection, evaluating the efficacy of therapeutic agents, and assisting with clinical diagnosis. Real-time PCR (qPCR) is the standard method for determining HTLV-1 proviral load (PVL). However, qPCR is dependent upon well-defined standards and calibration to a standard curve, precluding precise and accurate quantification particularly at low cell concentrations. In this study, we use a third generation PCR technique, droplet digital PCR (ddPCR), which allows for direct quantification of PVL. DNA samples are partitioned into thousands of nanoliter-sized droplets, amplified on a thermocycler, analyzed for fluorescent signal, and normalized to a housekeeping gene. Poisson distribution statistics are used to determine absolute copy numbers independently of a standard curve. We found that the intra-assay and inter-assay reliability of ddPCR are robust and more consistent than those of qPCR. Moreover, ddPCR yields similar patient PVL by quantifying different viral genes. Our preliminary data suggests ddPCR can also be used to examine viral gene expression, a key player in the pathogenesis of HAM/TSP and an important parameter to monitor during therapeutic interventions in this condition. The reliability and precision of ddPCR for HTLV-1 quantification should be widely acknowledged as it might be an important tool to further understand the clinical correlates of the viral load in the context of HAM/TSP and other HTLV-1 related conditions; however, further investigation is required to determine the efficacy of ddPCR for mRNA analysis.