Abstract
The continued resurgence of the COVID-19 pandemic with multiple variants underlines the need for diagnostics that are adaptable to the virus. We have developed toehold RNA-based sensors across the SARS-CoV-2 genome for direct and ultrasensitive detection of the virus and its prominent variants. Here, isothermal amplification of a fragment of SARS-CoV-2 RNA coupled with activation of our biosensors leads to a conformational switch in the sensor. This leads to translation of a reporter protein, for example, LacZ or nano-lantern that is easily detected using color/luminescence. By optimizing RNA amplification and biosensor design, we have generated a highly sensitive diagnostic assay that is capable of detecting as low as 100 copies of viral RNA with development of bright color. This is easily visualized by the human eye and quantifiable using spectrophotometry. Finally, this PHAsed NASBA-Translation Optical Method (PHANTOM) using our engineered RNA biosensors efficiently detects viral RNA in patient samples. This work presents a powerful and universally accessible strategy for detecting COVID-19 and variants. This strategy is adaptable to further viral evolution and brings RNA bioengineering center-stage.
Highlights
The COVID-19 pandemic has affected millions of people and caused severe disease, mortality, and disruption to human activity across the world
As a strategy for simple and specific SARS-CoV-2 detection that is compatible with a range of assay formats, we focused on direct detection of viral RNA fragments using an RNA biosensor approach
Each biosensor consists of a sensing region contiguous with a conserved stem-loop structure that contains a ribosome binding site (RBS) and a translation start site followed by a reporter gene (Fig 1A)
Summary
The COVID-19 pandemic has affected millions of people and caused severe disease, mortality, and disruption to human activity across the world. The COVID-19 disease is caused by a novel coronavirus SARS-CoV-2, belonging to the Betacoronavirus genus under the Coronaviridae family of viruses [1]. Because of the large numbers of potential infections, the high infectivity of the virus and the wide diversity in the clinical presentation of the SARS-CoV-2 infections, there is an ongoing need for reliable and efficient diagnostic methods. This is especially felt because a substantial portion of human subjects infected with SARS-CoV-2 are asymptomatic or show very mild symptoms but may still remain infectious. Detection strategies need to keep up with the evolving viral variants
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