General and Inexpensive Saliva-based Viral RNA Testing by Direct Imaging
Existing nucleic-acid-based COVID‑19 tests use enzymes to amplify low-copy number RNA to detectable levels. This adds cost, complexity and time to viral detection. In this proposal we will image fluorescently labeled probes hybridized to individual SARS-CoV-2 genomic RNA strands to enable a robust and highly sensitive test. The detection system involves three key steps: 1. Sample stabilization: As the assay does not require enzymes, robust stabilization of samples derived from saliva or nasal swabs is dramatically simplified. Detergents and mild denaturants will release viral RNA and homogenize any cell debris while serving to minimize virulence. 2. Hybridization and Washing: Probes within the stabilization solution will hybridize to viral RNA, brightly labeling it with quantum dots. Control RNA, such as human 18S RNA, will be similarly labelled but in a different colour channel. Sample preparation will be as simple as spitting into a sealable reagent container and shaking. A glass slide derivatized with viral and control probe oligonucleotides will then be introduced into the container and incubated for a defined time to immobilize the fluorescently tagged RNAs onto the slide as individual molecules. After hybridization, the slide will be rinsed stringently and placed into an imager. 3. Single molecule imaging. An inexpensive (target cost <$50) and easily mass-produced microscope will be designed to attach to a cell phone for single-molecule imaging. Images will be analyzed and uploaded to a medical site for analysis. Additional advantages over conventional tests include long-term storage and shipping for a test that is rapid (<15 min) and simple for point-of-care use. The test can be easily adapted to other RNA viruses, allowing a suite of tests to be rapidly deployed. This technology has the potential to revolutionize viral testing in remote locations, the home, and clinical settings to the benefit of all Canadians.