Project Summary The ongoing COVID-19/SARS-CoV-2 pandemic highlights the need for simple, rapid, and cost-effective testing for respiratory infections at the point-of-care, including physician’s offices, urgent-care settings, ambulatory procedural centers, and low-resource environments. The need is particularly notable for respiratory infections, such as COVID-19 and influenza, which can present with similar symptoms yet require distinct management strategies. With its high sensitivity and specificity, RT-PCR is the gold standard for the molecular diagnosis and differentiation among respiratory pathogens. Traditional RT-PCR workflow requires significant control over specimen contents and reaction conditions, with current methods requiring nucleic acid extraction prior to amplification and detection. The net result is increased complexity, cost, and/or turnaround time for diagnosis. In this context, we have observed in recent influenza studies that outstanding analytic performance characteristics can be achieved without RNA extraction, by applying our novel workflow and Adaptive PCR technology. Unlike traditional RT-PCR, Adaptive RT-PCR incorporates mirror-image L-DNA enantiomers— identical in sequence to PCR primers and targets—that modify cycling conditions to match the biochemical sample contents, thus eliminating the need to monitor reaction temperature. The direct monitoring of reaction conditions overcomes many of the limitations of traditional PCR, facilitating direct amplification within the original specimen matrix, simplifying instrument design, and enabling single-tube analyses. SARS-CoV-2 and influenza are both enveloped RNA viruses, with specimens collected in the same manner (i.e. nasopharyngeal swab) and using the same viral transport medium. Therefore, we hypothesize that we may eliminate RNA extraction for this virus, like we have done for influenza, by performing Adaptive RT-PCR directly on clinical specimens. We propose to enable a simplified methodology through Adaptive RT-PCR, creating diagnostics for COVID-19 and other respiratory pathogens without RNA extraction. As a collaboration between biomedical engineers and a COVID-19 diagnostic laboratory, we seek to develop a workflow and instrument that are simple-to-use, cost-effective, and suitable for point-of-care settings, tools that can rapidly inform treatment and management strategies. To achieve this goal, Aim 1 will evaluate the performance of RT- PCR directly – that is, without RNA extraction – using both traditional and Adaptive RT-PCR instrumentation. Aim 2 will develop multiplexed amplification reagents to create a sensitive and specific respiratory panel that detects SARS-CoV-2, four other viruses, two bacteria, and one control target. Ultimately, Aim 3 will design and fabricate a self-contained Adaptive RT-PCR instrument suitable for point-of-care settings, while validating this system using characterized human specimens in a CLIA-accredited lab environment. Comple...