Digital PCR (dPCR) technology has drawn attention in both the genomic research and clinical research communities for its ability to detect rare events (high sensitivity), be less prone to inhibition (high specificity), quantify without a standard curve (high precision), and exploit well-developed qPCR reagents. However, current dPCR platforms are slow to overtake the current qPCR gold standard due to their high consumable costs, excessively complicated workflows and incompatibility with automation. COMBiNATi's integrated single-instrument and micromolded consumable technology will deliver the first turnkey dPCR platform with efficient running cost for absolute nucleic acid quantification. In this study, we will exploit our integrated instrumentation to be the first ever to demonstrate single-color dPCR multiplexibility using High Resolution Melt (HRM) analysis of end-point dPCR partitions. For this Phase 1 SBIR project, the collaboration of COMBiNATi co-founder Dr. Megan Dueck and clinical HIV specialist Dr. Steve Yukl will strive to prove the feasibility of a novel multiplexed, single-color HRM dPCR assay for studies involving the latent HIV reservoir. Understanding the mechanisms leading up to HIV latency is paramount to finding an ultimate HIV cure. During the 9 month Phase 1 SBIR project, COMBiNATi co-founder Dr. Megan Dueck in collaboration with Dr. Steve Yukl will complete the following key deliverables: 1. Design and prototype a large field of view, high sensitivity optical module for real- time imaging of individual dPCR reaction partitions during incremental increase in temperature. As the first single-instrument dPCR platform, COMBiNATi strives to be the first to show multiplexed, single-color nucleic acid quantification using HRM analysis of endpoint dPCR partitions. Toward this end we will build an optical module capable of generating target-specific HRM curves for individual end-point dPCR partitions. 2. Develop a single-color, multiplexed HRM dPCR assay for ?transcription profiling? of the latent HIV reservoir. Our novel HRM dPCR multiplexing strategy will be benchmarked using a panel of 3 pre-validated dPCR assays that quantify markers for different stages of HIV transcriptional blockade. We hope to ultimately develop a robust single-color dPCR assay for examining mechanisms of HIV latency.
Despite its promise, digital PCR (dPCR) is slow to overtake the traditional quantitative PCR (qPCR) gold-standard as the ?go-to? technology for targeted nucleic acid quantification. COMBiNATi strives to accelerate this adoption with our fully-integrated, streamlined and cost- effective dPCR platform. In this study, we aim to be the first ever to achieve single-color dPCR multiplexibility using High Resolution Melt (HRM) analysis of end-point dPCR partitions. To benchmark this novel multiplexing strategy we will develop a method for measuring multiple markers for HIV transcription blockade within the same reaction. The assay we develop will be a valuable tool for studies involving the latent HIV reservoir.
