The coronavirus disease 2019 (COVID-19) pandemic caused millions of deaths and serious economic disruptions, also boosted the unprecedented development of novel nucleic acid detection methods. Although polymerase chain reaction (PCR) is the golden-standard and has been widely used for practical nucleic acid detection, isothermal amplification strategies capable of rapid, inexpensive, and accurate nucleic acid detection also provide new options for large-scale pathogen detection, disease diagnosis, and genotyping. Here we are going to describe an assay development journey from a simple COVID-19 detection assay to a genotyping strategy and eventually to a droplet-based amplification-free assay.
First, we described a highly sensitive multicomponent XNA-based nucleic acid detection platform that combines analyte preamplification with X10–23 mediated catalysis to detect the viral pathogen responsible for COVID-19. It is termed RNA-Encoded Viral Nucleic Acid Analyte Reporter (REVEALR), functions with a detection limit of ≤20 aM (∼10 copies/μL) using conventional fluorescence and paper-based lateral flow readout modalities. With a total assay time of 1 h, REVEALR provides a convenient nucleic acid alternative to equivalent CRISPR-based approaches, which have become popular methods for SARS-CoV-2 detection. The assay shows no cross-reactivity for other in vitro transcribed respiratory viral RNAs and functions with perfect accuracy against COVID-19 patient-derived clinical samples.
Second, we explained how we design REVEALR into a novel genotyping assay that detects single-base mismatches corresponding to each of the major SARS-CoV-2 strains found in the United States. Of 34 sequence-verified patient samples collected in early, mid, and late 2021 at the UCI Medical Center in Orange, California, REVEALR accurately identified the correct variant. The assay, which is programmable and amenable to multiplexing, offers an important new approach to personalized diagnostics.
Third, we talked about an improved REVEALR platform, termed digital droplet REVEALR (ddREVEALR), that can achieve direct viral detection and absolute quantitation utilizing a signal amplification strategy that relies on 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 nasopharyngeal swabs collected at UCI Medical Center in Orange, California. We suggest that the combination of amplification-free and protein-free analysis makes ddREVEALR a promising approach for direct viral RNA detection of clinical samples.
Finally, we summarized the developed DNAzyme-based nucleic acid detection methods, offered some alternatives, compared the DNAzyme-based platforms with CRISPR based platforms, and gave insight on potential future directions to further elevate the REVEALR system.