The goal of this Phase I SBIR proposal is to develop a 15 minute multiplex point-of-care (POC) molecular testfor the qualitative detection of COVID-19 and influenza A/B. As of September 2021, the US has had over 39.5million confirmed cases of COVID-19 and over 642,000K deaths. Additionally, influenza viruses account for over 200,000 hospitalizations and 30,000-50,000 deaths in the US each year. As SARS-CoV-2 and influenzainfections can hardly be differentiated by symptoms alone, a single test that can diagnose illness caused bySARS-CoV-2 and influenza viruses will give public health officials information they need in their efforts to controlthe spread of these important viruses of concern. Our approach leverages rapid sample preparation (1 minhands-on-time and 2.5 minute of heat inactivation) and the rapid gold-standard real-time RT-PCR (12 minute,40-cycle) to amplify the targeted viral genome. This test can be utilized in multiple settings where actionable testresults are needed to make informed treatment decisions quickly. We will demonstrate that our test can deliverrapid results with a similar level of performance as those obtained in reference labs using nasopharyngeal swabsand contrived saliva samples. Our approach has been tested using SARS-CoV-2 and influenza positive clinicalspecimens in viral transport media. We plan to achieve rapid multiplex RT-PCR using shuttle PCR with thin-filmreactors. We can enhance PCR speed and efficiency by using vibration to enhance reagent mass-transport whileavoiding the "extreme PCR" approach that needs 10 to 20 times more concentrated reagents (i.e., additionalexpense) to speed up the reaction. The result from spatial multiplexed fluorescence probe-based RT-PCR canbe imaged and analyzed by on-board electronics. The RT-PCR test takes ~8 minutes to complete 40 cycles after2.5 min of sample heat inactivation and 3 min of reverse-transcription step, and some high positive results canbe obtained in as early as 6 minutes. Our approach will be much more sensitive than antigen-based approachesand faster than isothermal amplification approaches, such as loop mediated isothermal amplification (LAMP).The diagnostic utility of the molecular assay will be demonstrated by comparing its robustness, speed, sensitivity,and specificity with current molecular assays. In Phase I, we will work with our collaborator to validate our deviceand test using clinical specimens. If successfully developed, this compact and minimal instrumentation approachwill be simple to perform and inexpensive enough for all sizes of primary-care physician's offices, nursing homes,pharmacies, community health clinics, and even at-home consumers to adopt the platform.
Public Health Relevance Statement: PROJECT NARRATIVE A low cost, rapid, multiplex point-of-care molecular diagnostic test for SARS-CoV-2 and influenza A and B is greatly needed at the local level. Our proposed 15-min rapid assay will ultimately detect the presence of SARS- CoV-2 and influenza in minimally altered specimens using a gold standard RT-PCR assay. This test can be performed in point-of-care and at-home settings where existing instrumentation-based molecular tests would be too costly to implement.
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