Pdot-Enabled Point-of-Care Digital PCR for Sensitive Detection of SARS-CoV-2

Project Summary Current SARS-CoV-2 molecular diagnosis tests mostly are based on real-time quantitative PCR (qPCR). Digital PCR is a next-generation PCR technology based on limiting dilution, end-point PCR, and Poisson statistics. Digital PCR transforms the exponential, analog nature of qPCR quantification into a linear, digital signal quantification. Compared to qPCR, digital PCR offers several important advantages, including its ability to provide absolute quantification, tolerance to inhibitors/contaminants, and its high sensitivity. Two recent studies showed digital PCR has improved sensitivity for picking up COVID-19 that qPCR had missed. In addition, for some patients, results on SARS-CoV-2 infections from qPCR varied from day to day. But unlike qPCR, digital PCR showed consistent reproducible results. Overall, digital PCR was shown to reduce significantly false-negative results, which is particularly useful for diagnosing early or asymptomatic infections or for testing convalescent patients before discharge. Current fluorescent probes used in qPCR, such as FAM (a fluorescein dye), do not have the brightness to be visualized with a cell-phone type camera. To address this issue and to enable simple cell-phone readout of digital PCR results in a point-of-care setting, this project aims to develop Pdots into ultrabright probes for digital PCR assays that can provide sensitive detection of SARS-CoV-2.Project Narrative Current SARS-CoV-2 molecular diagnosis tests mostly are based on real-time PCR (qPCR). Digital PCR is a quantification method based on end-point PCR and Poisson statistics, and is more sensitive and precise that qPCR, especially at low copy numbers, which is particularly relevant in detecting SARS-CoV-2 in Covid-19 patients with early or asymptomatic infections or for testing convalescent patients before discharge. This proposal aims to develop a Pdot- enabled digital PCR assay for the sensitive detection of SARS-CoV-2.