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Title & authors Abstract Conflict of interest statement Figures References Publication types MeSH terms Substances Grants and funding LinkOut - more resources Full text links CiteDisplay options Display options Format AbstractPubMedPMID
Abstract
An outbreak of betacoronavirus severe acute respiratory syndrome (SARS)-CoV-2 began in Wuhan, China in December 2019. COVID-19, the disease associated with SARS-CoV-2 infection, rapidly spread to produce a global pandemic. We report development of a rapid (<40 min), easy-to-implement and accurate CRISPR-Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts. We validated our method using contrived reference samples and clinical samples from patients in the United States, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US Centers for Disease Control and Prevention SARS-CoV-2 real-time RT-PCR assay, with 95% positive predictive agreement and 100% negative predictive agreement.
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COMPETING INTERESTS
CYC is the director of the UCSF-Abbott Viral Diagnostics and Discovery Center (VDDC), receives research support funding from Abbott Laboratories, and is on the Scientific Advisory Board of Mammoth Biosciences, Inc. JSC is a co-founder of Mammoth Biosciences, Inc. JPB, CLF, JS and XM are employees of Mammoth Biosciences, Inc. CYC, JPB, XD, CLF, JS, XM and JSC are co-inventors of CRISPR-related technologies.
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Figure 1.
A CRISPR-Cas12 based assay for…
Figure 1.
A CRISPR-Cas12 based assay for detection of SARS-CoV-2. (a) Genome map showing primers,…
Figure 1. A CRISPR-Cas12 based assay for detection of SARS-CoV-2. (a) Genome map showing primers, probes and gRNAs. Visualization of primers and probes on the SARS-CoV-2 genome. RT-LAMP primers are indicated by black rectangles, the binding position of the F1c and B1c half of the FIP primer (grey) is represented by a striped rectangle with dashed borders. (b) gRNA specificity. Cas12 gRNAs are programmed to specifically target SARS-CoV-2 or broadly detect related coronavirus strains. The N gene gRNA used in the assay (left) is specific for SARS-CoV-2, whereas the E gene gRNA is able to detect 3 SARS-like coronavirus strains (right). A separate N gene gRNA designed to target SARS-CoV and a bat coronavirus fails to detect SARS-CoV-2 (middle). (c) Minimum equipment needed to run protocol. With appropriate biosafety level 2 requirements, the minimum equipment required to run the protocol includes Eppendorf tubes with reagents, heat blocks or water bath (37°C and 62°C), nuclease-free water, pipettes and tips, and lateral flow strips. (d) Schematic of SARS-CoV-2 DETECTR workflow. Conventional RNA extraction can be used as an input to DETECTR (LAMP pre-amplification and Cas12-based detection for E gene, N gene and RNase P), which is visualized by a fluorescent reader or lateral flow strip. (e) Lateral flow strip assay readout. A positive result requires detection of at least one of the two SARS-CoV-2 viral gene targets (N gene or E gene, as indicated in the interpretation matrix).
Figure 2.
Detection of SARS-CoV-2 in contrived…
Figure 2.
Detection of SARS-CoV-2 in contrived and clinical nasopharyngeal or oropharyngeal swab samples. (a)…
Figure 2. Detection of SARS-CoV-2 in contrived and clinical nasopharyngeal or oropharyngeal swab samples. (a) Schematic of DETECTR coupled with lateral flow readout. The intact FAM-biotinylated reporter molecule flows to the control capture line. Upon recognition of the matching target, the Cas-gRNA complex cleaves the reporter molecule, which flows to the target capture line. (b-c) Comparison of fluorescence to lateral flow. (b) Fluorescence signal of LbCas12a detection assay on RT-LAMP amplicon for SARS-CoV-2 N gene saturates within 10 min. RT-LAMP amplicon generated from 2 µL of 5 fM or 0 fM SARS-CoV-2 N gene IVT RNA by amplifying at 62°C for 20 min. Error bars: mean±SD.(c) LbCas12a on the same RT-LAMP amplicon produces visible signal through lateral flow assay within 5 min. (d) Limit of detection for CDC qPCR and DETECTR. Ct values using the CDC qPCR assay (n=3) and fluorescence values using SARS-CoV-2 DETECTR (n=6) using SARS-CoV-2 N2 gene IVT-RNA. Representative lateral flow results for the assay shown for 0 copies/µL and 10 copies/µL. (e) Patient sample DETECTR data. Clinical samples from 6 patients with COVID-19 infection (n=11, 5 replicates) and 12 patients infected with influenza or one of the 4 seasonal coronaviruses (HCoV-229E, HCoV-HKU1, HCoV-NL63, HCoV-OC43) (n=12) were analyzed using SARS-CoV-2 DETECTR. Signal intensities from lateral flow strips were quantified using ImageJ and normalized to the highest value within the N gene, E gene or RNase P set, with a positive threshold set at five standard deviations above background. The final determination for SARS-CoV-2 test results was based on the interpretation matrix in Fig. 1e, with results indicated above the heat map. (f) Lateral flow strips showing SARS-CoV-2 DETECTR assay results. Two replicate assays were performed using 2 µL of extracted RNA for each reaction (titer 12 copies/µL). Positive controls used IVT RNA for SARS-CoV-2 targets and total human RNA for RNase P. LbCas12a detection assays were run on lateral flow strips (TwistDx) and imaged after 3 min. (g) Performance characteristics of the fluorescent SARS-CoV-2 DETECTR assay. 83 clinical samples (41 COVID-19 positive, 42 negative) were evaluated using the fluorescent version of the SARS-CoV-2 DETECTR assay (Supp. Fig. 7 a, c, d). One sample (COVID19–3) was omitted due to failing assay quality control. Positive and negative calls are based on criteria described in Fig. 1e. Abbreviations: fM, femtomolar; NTC, no-template control; FLUA, influenza A virus; FLUB, influenza B virus; HCoV, human coronavirus; PPA, positive predictive agreement; NPA, negative predictive agreement. See this image and copyright information in PMC
References
Wang C, Horby PW, Hayden FG & Gao GF A novel coronavirus outbreak of global health concern. Lancet 395, 470–473, doi:10.1016/S0140-6736(20)30185-9 (2020). - DOI - PMC - PubMed
Zhu N et al. A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med 382, 727–733, doi:10.1056/NEJMoa2001017 (2020). - DOI - PMC - PubMed
World Health Organization. Coronavirus disease 2019 (COVID-19) Situation Report - 44 8 (World Health Organization, Geneva, Switzerland, 2020).
Bai Y et al. Presumed Asymptomatic Carrier Transmission of COVID-19. JAMA, doi:10.1001/jama.2020.2565 (2020). - DOI - PMC - PubMed
Rothe C et al. Transmission of 2019-nCoV Infection from an Asymptomatic Contact in Germany. N Engl J Med 382, 970–971, doi:10.1056/NEJMc2001468 (2020). - DOI - PMC - PubMed
Show all 23 references
METHODS-ONLY REFERENCES
Elbe S & Buckland-Merrett G Data, disease and diplomacy: GISAID’s innovative contribution to global health. Glob Chall 1, 33–46, doi:10.1002/gch2.1018 (2017). - DOI - PMC - PubMed
Curtis KA et al. A multiplexed RT-LAMP assay for detection of group M HIV-1 in plasma or whole blood. J. Virol. Methods 255, 91–97, doi:10.1016/j.jviromet.2018.02.012 (2018). - DOI - PubMed
Waterhouse AM, et al. Jalview Version 2 – a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191, doi: 10.1093/bioinformatics/btp033 (2009). - DOI - PMC - PubMed
McWilliam H, et al. Analysis Tool Web Services from the EMBL-EBI. Nucleic Acids Research 41, doi:10.1093/nar/gkt376 (2013). - DOI - PMC - PubMed
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R01-HL105704/U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/International
R01 HL105704/HL/NHLBI NIH HHS/United States
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