Design of a Rapid and Reversible Fluorescence Assay to Detect Covid-19 and other Pathogens

Abstract

We describe a rapid and reusable biophysical method to assay COVID-19.  The method uses fluorescent sensors (i.e., molecular beacons) designed to detect a specific RNA sequence from COVID-19 but is general to any RNA of interest.  The assay can be used concurrently with an internal control without the need for amplification.  Molecular beacons are stem-loop structures in which a ~10 nucleotide loop region has the complementary sequence of a region of the target RNA, and a fluorophore and quencher are placed on the 5’ and 3’ ends of the stem. The energy of hybridization of the loop with its target is designed to be greater than the hybridization energy of the energy of the stem so that when the beacon encounters its target RNA, the structure opens resulting in dequenching of the fluorophore.  Here, we designed a beacon to different COVID-19 variants that is completely quenched in its native form and undergoes a 50-fold increase in fluorescence when exposed to nanomolar amounts of synthetic viral oligonucleotide.  No changes in intensity are seen when a control RNA (hGAPDH) is added. This increase in fluorescence with beacon opening can be completely reversed upon addition of single stranded DNA complementary to COVID-19 beacon loop region.  Beacons can be attached to an inert matrix allowing their use and reuse in concentrated form and can be made from morphilino oligonucleotides that are resistant to RNases. We present an analysis of the parameters that will allow the development of test strips to detect virus in aerosol, body fluids and community waste.