Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2

Abstract

The COVID-19 pandemic has highlighted that new diagnostic technologies are essential for controlling disease transmission. Here, we develop SHINE (Streamlined Highlighting of Infections to Navigate Epidemics), a sensitive and specific diagnostic tool that can detect SARS-CoV-2 RNA from unextracted samples. We identify the optimal conditions to allow RPA-based amplification and Cas13-based detection to occur in a single step, simplifying assay preparation and reducing run-time. We improve HUDSON to rapidly inactivate viruses in nasopharyngeal swabs and saliva in 10 min. 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-qPCR with a sample-to-answer time of 50 min. SHINE has the potential to be used outside of hospitals and clinical laboratories, greatly enhancing diagnostic capabilities.

Fig. 1. Initial assay development for SHERLOCK-based SARS-CoV-2 detection.

a Schematic of single-step SHERLOCK assays using extracted RNA with a fluorescent or colorimetric readout. RT-RPA reverse transcriptase-recombinase polymerase amplification, C control line, T test line. b Schematic of the SARS-CoV-2 genome and SHERLOCK assay location. Sequence conservation across the primer and crRNA-binding sites for publicly available SARS-CoV-2 genomes (see “Methods” for details). Text denotes nucleotide position with lowest percent conservation across the assay location. ORF open reading frame, T7pro T7 polymerase promoter; narrow rectangles, untranslated regions. c Colorimetric detection of synthetic RNA using two-step SHERLOCK after 30 min. NTC_r non-template control introduced in RPA, NTC_d non-template control introduced in detection, T test line, C control line. d Background-subtracted fluorescence of the two-step and original single-step SHERLOCK protocols using synthetic SARS-CoV-2 RNA after 3 h. The 1-h timepoint from this experiment is shown in Fig. 2e. NTC non-template control introduced in RPA. Center = mean and error bars = s.d. for 3 technical replicates. For b–d, source data are provided as a Source data file.

Fig. 2. Optimization of the single-step SHERLOCK reaction.

a Background-subtracted fluorescence of Cas13-based detection with synthetic RNA, reverse transcriptase, and RPA primers (but no RPA enzymes) after 3 h. b Single-step SHERLOCK normalized fluorescence using various buffering conditions after 3 h. c Background-subtracted fluorescence of single-step SHERLOCK with synthetic RNA and variable RPA forward and reverse primer concentrations after 3 h. d Single-step SHERLOCK normalized fluorescence over time using two different fluorescent reporters (left) and two different reverse transcriptases (right). e Background-subtracted fluorescence of the original single-step and optimized single-step SHERLOCK with synthetic RNA after 1 h. Data from the 3-h timepoint from this experiment are shown in Fig. 1d. f Colorimetric detection of synthetic RNA input using optimized single-step SHERLOCK after 3 h. Max maximum test band intensity, 5698.4 a.u., Min minimum test band intensity, 104.4 a.u. g Optimized single-step SHERLOCK background-subtracted fluorescence using RNA extracted from patient samples after 1 h. h Concordance between SHERLOCK and RT-qPCR for 7 patient samples and 4 controls. For c, e, see “Methods” for details about normalized fluorescence calculations. For b, d, f, g, NTC non-template control. For a, c, center = mean for 2 technical replicates. For d–f, center = mean and error bars = s.d. for 3 technical replicates. For b, d, RNA input at 10⁴ cp/μL. For a–e, g, source data are provided as a Source data file.

Fig. 3. SARS-CoV-2 detection from unextracted samples using SHINE.

a Schematic of SHINE, which streamlines SARS-CoV-2 detection by using HUDSON to inactivate samples and single-step SHERLOCK to detect viral RNA with an in-tube fluorescent or colorimetric readout. Times suggested incubation times, C control line, T test line. b Measurement of RNase activity using RNaseAlert after 30 min at room temperature from treated or untreated universal viral transport medium (UTM), saliva, and phosphate-buffered saline (PBS). c SARS-CoV-2 RNA detection in UTM using SHINE with the in-tube fluorescence readout after 1 h. d SARS-CoV-2 RNA detection in saliva using SHINE with the in-tube fluorescence readout after 1 h. e Schematic of the companion smartphone application for quantitatively analyzing in-tube fluorescence and reporting binary outcomes of SARS-CoV-2 detection. f Colorimetric detection of SARS-CoV-2 RNA in unextracted patient NP swabs using SHINE after 1 h. g SARS-CoV-2 detection from 50 unextracted patient samples using SHINE and smartphone application quantification of in-tube fluorescence after 40 min. Threshold line plotted as mean readout value for controls plus 3 standard deviations. h Concordance table between SHINE and RT-qPCR for 50 patient samples. For b, center = mean for 2 technical replicates. For b, g, source data are provided as a Source data file.