Equipment-free detection of SARS-CoV-2 and Variants of Concern using Cas13

Abstract

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.

Fig. 1 |. Increasing the ease-of-use and deployability of SHINE.

a, RNase activity in nasal fluid mixed with universal viral transport medium (UTM) untreated or treated with FastAmp Lysis reagent supplemented with RNase inhibitor (inh.) or treated with HUDSON (a heat- and chemical- treatment). Activity measured using RNaseAlert at room temperature (RT) for 30 minutes. b, SARS-CoV-2 seedstock titer without treatment or after being incubated with lysis reagent (+5% RNase inh.) at RT for 5 minutes, 20 minutes or 20 minutes plus 10 minutes at 65°C. ***, infection not detected; PFU, plaque forming units. c, SHINE fluorescence with different proportions of inactivated sample input (i.e. FastAmp lysis reagent, RNase inh. and UTM) after a 90 minute incubation. Base*, baseline (i.e. no inactivated sample added). d, Schematic of the advantages of lyophilizing SHINE. e, SHINE fluorescence on synthetic RNA target (10⁴ copies/μL) before and after lyophilization using different buffers. Fluorescence measured after 90 minutes. For buffer composition, see Methods. f, SHINE fluorescence after a 90 minute incubation using lyophilized (LYO) reagents stored at RT, 4°C or −20°C over time. Target concentration: 10⁴ copies/μL. g, Fluorescence kinetics for SHINEv1 and SHINEv2 using synthetic RNA targets; NTC, no target control. h, Lateral flow detection of SARS-CoV-2 RNA in lysis buffer treated viral seedstocks using SHINEv2, after a 90 minute incubation. C = control band; T = test band. i, Determination of analytical limit of detection with 20 replicates of SHINEv2 at different concentrations of SARS-CoV-2 RNA from lysis reagent treated contrived samples. Incubated for 90 minutes. For (a,e,g), center = mean and error bars = standard deviation (s.d.) for 3 technical replicates. In c, the heatmap values represent the mean for 3 technical replicates. For f, center = mean and error bars = s.d. for 3 biological replicates with 3 technical replicates each.

Fig. 2 |. Performance of SHINEv2 on clinical samples.

a, Schematic of side-by-side clinical sample testing using SHINEv2, BinaxNow, CareStart and RT-qPCR. NP, nasopharyngeal swab. b, SHINEv2, BinaxNow and CareStart test results for a subset of clinical NP swab samples with different Ct values (CDC EUA N1 RT-qPCR). C = control band; T = test band. No Amp., no amplification detected. For all test results, see Supplementary Fig. 8–10. c, Side-by-side clinical performance of SHINEv2, BinaxNow and CareStart versus RT-qPCR. d, Positive and negative test results for SHINEv2, BinaxNow and CareStart tests for RT-qPCR-positive clinical samples relative to viral RNA concentration and Ct value.

Fig. 3 |. Development of SHINEv2 assays for the detection of SARS-CoV-2 VOCs.

a, Schematic of Cas13a-based detection of mutations in SARS-CoV-2 using a fluorescent readout; anc, ancestral; der, derived. (b,c), SHINE fluorescence of the ancestral and derived crRNAs for the (b) 69/70 deletion assay and (c) on synthetic RNA targets after 90 minutes; NTC, no target control. d, Identification of SARS-CoV-2 VOCs using SHINE fluorescence on full-genome synthetic RNA standards and RNA extracted from viral seedstocks; target RNA concentration: 10⁴ copies/μL. e, Mean fluorescence of 69/70 SHINEv2 assay on SARS-CoV-2 RNA extracted from clinical samples, after 90 minutes. f, Discrimination of SARS-CoV-2 VOCs using SHINE fluorescence of Delta assays on full-genome synthetic RNA standards, after 90 minutes. Target RNA concentration: 10⁴ genomes/μL. (g,h), Colorimetric lateral flow based detection of full-synthetic RNA standards using the (g) 156 – 158 and (h) SHINEv2 assays. SHINEv2 incubation time: 90 minutes. NTC, no target control. T, test line; C, control line. For b and c, center = mean and error bars = s.d. for 3 technical replicates. In d,e and f, the heatmap values represent the mean for 3 technical replicates.

Fig. 4 |. Enhancing the accessibility of SHINEv2.

a, SHINE fluorescence of the RNase P SHINEv2 assay on synthetic DNA target after 90 minutes; NTC, no target control. b, Lateral flow based detection of SARS-CoV-2 RNA using SHINEv2 with different polyethylene glycol (i.e. PEG) compositions; with or without dilution after a 90 minute incubation. NTC, no target control. c, Lateral flow based SHINEv2 detection of SARS-CoV-2 RNA after a 90 minute incubation in a heat block or using body heat (underarm incubation). NTC, no target control. d, SHINE fluorescence on SARS-CoV-2 RNA after 90 minutes at 37°C or 25°C; NTC, no target control. For d, center = mean and error bars = s.d. for 3 technical replicates.