UC Irvine

Since the emergence of the coronavirus SARS-CoV-2 in 2019, unprecedented rates of transmission coupled with alarmingly high mortality rates have remained the focus of the current healthcare climate. Despite the development of vaccines designed to combat virulence and the severity of the symptoms, the global healthcare crisis has continued through to 2022. Additionally, existing golden standard RNA-based diagnostics such as RT-PCR require expensive instrumentation, careful handling and extensive training, services that may not be immediately available to some vulnerable communities and limit their response during an epidemic. These issues have become increasingly evident with continued transmission stemming from mutation of the virus, resulting in a multitude of different lineages with varying degrees of infectivity, endangering the efficacy of existing solutions. To address these shortcomings in the global response, an RNA-based diagnostic tool has been developed that provides an isothermal and easy-to-handle option for screening samples for the presence of the Wuhan strain of the SARS-CoV-2 virus (Wuhan-Hu-1) and differentiation from the UK (B.1.1.7) variant. In this work, two key steps have been incorporated for detection of regions of interest: 1) upstream isothermal amplification of key regions using Reverse Transcription-Recombinase Polymerase Amplification (RT-RPA), and 2) a proof-read step using duplex-specific nuclease that mediates further signal amplification only in the presence of a perfect DNA:RNA duplex. We further demonstrate compatibility with alternative low-resource signal enhancement strategies with a signal concentration method utilizing electro-osmotic concentration of bead-based fluorescence and detection on lateral flow nitrocellulose strips. The potential of this novel nuclease-based fluorescent assay is demonstrated as a user-friendly alternative for screening between relevant SARS-CoV-2 strains through detection of key regions within the ORF1a and S genes necessary to distinguish between the aforementioned strains (Wuhan-Hu-1, B.1.1.7).