Amplification-Free COVID-19 Detection by Digital Droplet REVEALR

Abstract

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, exposed a pressing need for new public health tools for pathogen detection, disease diagnosis, and viral genotyping. REVEALR (RNA-encoded viral nucleic acid analyte reporter) is an isothermal DNAzyme-based point-of-care diagnostic that functions with a detection limit of ∼10 copies/μL when coupled with a preamplification step and can be utilized for viral genotyping of SARS-CoV-2 variants of concern through base pair mismatch recognition in a competitive binding format. Here, we describe an advanced REVEALR platform, termed digital droplet REVEALR (ddREVEALR), that can achieve direct viral detection and absolute sample quantitation utilizing a signal amplification strategy that relies on chemical modifications, DNAzyme multiplexing, and volume compression. Using an AI-assisted image-based readout, ddREVEALR was found to achieve 95% positive predictive agreement from a set of 20 nasal pharyngeal swabs collected at UCI Medical Center in Orange, California. We propose that the combination of amplification-free and protein-free analysis makes ddREVEALR a promising application for direct viral RNA detection of clinical samples.

Engineering Gene-Specific DNAzymes for Accessible and Multiplexed Nucleic Acid Testing

“…To compensate for the reduction of activity, Chaput and colleagues introduced chemical modifications to MNAzymes, which successfully enhanced overall assay performances. A further integration with recombinase polymerase amplification (RPA), sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its mutants were achieved in clinical samples. − Although chemical modification could effectively rescue the split DNAzymes, it increases the upfront cost and accessibility of the test. An ideal engineering approach to achieve target-specific DNAzymes in a cost-effective manner should keep its integrity to ensure maximal enzymatic activity.…”