The widespread transmission and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) call for rapid nucleic acid diagnostics that are easy to use outside of centralized clinical laboratories. Here we report the development and performance benchmarking of Cas13-based nucleic acid assays leveraging lyophilised reagents and fast sample inactivation at ambient temperature. The assays, which we named SHINEv.2 (for 'streamlined highlighting of infections to navigate epidemics, version 2'), simplify the previously reported RNA-extraction-free SHINEv.1 technology by eliminating heating steps and the need for cold storage of the reagents. SHINEv.2 detected SARS-CoV-2 in nasopharyngeal samples with 90.5% sensitivity and 100% specificity (benchmarked against the reverse transcription quantitative polymerase chain reaction) in less than 90 min, using lateral-flow technology and incubation in a heat block at 37 °C. SHINEv.2 also allows for the visual discrimination of the Alpha, Beta, Gamma, Delta and Omicron SARS-CoV-2 variants, and can be run without performance losses by using body heat. Accurate, easy-to-use and equipment-free nucleic acid assays could facilitate wider testing for SARS-CoV-2 and other pathogens in point-of-care and at-home settings.

The COVID-19 pandemic, and the recent rise and widespread transmission of SARS-CoV-2 Variants of Concern (VOCs), have demonstrated the need for ubiquitous nucleic acid testing outside of centralized clinical laboratories. Here, we develop SHINEv2, a Cas13-based nucleic acid diagnostic that combines quick and ambient temperature sample processing and lyophilized reagents to greatly simplify the test procedure and assay distribution. We benchmarked a SHINEv2 assay for SARS-CoV-2 detection against state-of-the-art antigen-capture tests using 96 patient samples, demonstrating 50-fold greater sensitivity and 100% specificity. We designed SHINEv2 assays for discriminating the Alpha, Beta, Gamma and Delta VOCs, which can be read out visually using lateral flow technology. We further demonstrate that our assays can be performed without any equipment in less than 90 minutes. SHINEv2 represents an important advance towards rapid nucleic acid tests that can be performed in any location.

The high disease burden of influenza virus poses a significant threat to human health and requires better methods to rapidly detect its many circulating species, subtypes, and variants. No current diagnostic technology meets the combined critical needs for a rapid, sensitive, specific, and cost-effective method for point-of-need (PON) influenza detection and discrimination with minimal equipment requirements. Here, we introduce such a method using SHINE (Streamlined Highlighting of Infections to Navigate Epidemics), a CRISPR-based RNA detection platform. We develop and validate four SHINE assays for the detection and differentiation of clinically relevant influenza species (A and B) and subtypes (H1N1 and H3N2). These optimized assays achieve 100% concordance with reverse-transcriptase real-time polymerase chain reaction (RT-qPCR) when tested on clinical samples. We also created duplex Cas12/Cas13 SHINE assays to simultaneously detect two targets and demonstrate its use in discriminating two alleles of an oseltamivir resistance (H275Y) mutation as well as to detect influenza A and human RNAse P, as a built-in internal control. Our assays have the potential to expand influenza detection outside of clinical laboratories in order to enhance influenza diagnosis and surveillance.