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The well-recognized genome editing ability of the CRISPR-Cas system has triggered significant advances in CRISPR diagnostics. This has prompted an interest in developing new biosensing applications for nucleic acid detection. Recently, such applications have been engineered for detection of SARS-CoV-2. Increased demand for testing and consumables of RT-qPCR assays has led to the use of alternate testing options in some cases. Here we evaluate the accuracy and performance of a novel fluorescence based assay that received EUA authorization from the FDA for detecting SARS-CoV-2 in clinical samples. The Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) technology forms the basis of the Sherlock CRISPR SARS-CoV-2 kit using the CRISPR-Cas13a system. Our experimental strategy included selection of COVID-19 patient samples from previously validated RT-qPCR assays. Positive samples were selected based on a broad range of cycle thresholds. A total of 50 COVID-19 patient samples were correctly diagnosed with 100% accuracy (relative fluorescence ratios: N gene 95% CI 23.2-36.3, ORF1ab gene 95% CI 27.6- 45.4). All controls, including RNase P, showed expected findings. Overall ratios were robustly distinct between positive and negative cases relative to the pre-established 5-fold change in fluorescence read output. We have evaluated the accuracy of detecting conserved targets of SARS-CoV-2 across a range of viral loads using the SHERLOCK CRISPR collateral detection reaction in a clinical setting. These findings demonstrate encouraging results, especially at a time when COVID-19 clinical diagnosis is in high demand; often with limited resources. This approach highlights new thinking in infectious disease identification and can be expanded to measure nucleic acids in other clinical isolates.
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10.1101/2020.12.17.20228593
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document_parses/pdf_json/0a272df911be3e96ee0343bf7c5462c8ca0ecd3b.json
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CRISPR-cas13 enzymology rapidly detects SARS-CoV-2 fragments in a clinical setting
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