The emergence and outbreak of SARS-CoV-2, the causative agent of COVID-19, has rapidly become a global concern and has highlighted the need for fast, sensitive, and specific tools to surveil circulating viruses. Here we provide assay designs and experimental resources, for use with CRISPR-based nucleic acid detection, that could be valuable for ongoing surveillance. We provide assay designs for detection of 67 viral species and subspecies, including: SARS-CoV-2, phylogenetically-related viruses, and viruses with similar clinical presentation. The designs are outputs of algorithms that we are developing for rapidly designing nucleic acid detection assays that are comprehensive across genomic diversity and predicted to be highly sensitive and specific. Of our design set, we experimentally screened 4 SARS-CoV-2 designs with a CRISPR-Cas13 detection system and then extensively tested the highest-performing SARS-CoV-2 assay. We demonstrate the sensitivity and speed of this assay using synthetic targets with fluorescent and lateral flow detection. Moreover, our provided protocol can be extended for testing the other 66 provided designs. Assay designs are available at https://adapt.sabetilab.org/ .
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 has highlighted that new diagnostic technologies are essential for controlling disease transmission. Here, we develop SHINE (SHERLOCK and HUDSON Integration to Navigate Epidemics), a sensitive and specific integrated diagnostic tool that can detect SARS-CoV-2 RNA from unextracted samples. We combine the steps of SHERLOCK into a single-step reaction and optimize HUDSON to accelerate viral inactivation in nasopharyngeal swabs and saliva. SHINE's results can be visualized with an in-tube fluorescent readout -reducing contamination risk as amplification reaction tubes remain sealed -and interpreted by a companion smartphone application. We validate SHINE on 50 nasopharyngeal patient samples, demonstrating 90% sensitivity and 100% specificity compared to RT-PCR with a sample-to-answer time of 50 minutes. SHINE has the potential to be used outside of hospitals and clinical laboratories, greatly enhancing diagnostic capabilities.
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.
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