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Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy.

ABSTRACT: The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved ∼100 copies/μL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2.

SUBMITTER: Fozouni P

PROVIDER: S-EPMC7834310 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Abstract CRISPR (Clustered regularly interspaced short palindromic repeats)‐based diagnostic technologies have emerged as a promising alternative to accelerate delivery of SARS‐CoV‐2 molecular detection at the point of need. However, efficient translation of CRISPR‐diagnostic technologies to field application is still hampered by dependence on target amplification and by reliance on fluorescence‐based results readout. Herein, an amplification‐free CRISPR/Cas12a‐based diagnostic technology for SARS‐CoV‐2 RNA detection is presented using a smartphone camera for results readout. This method, termed Cellphone‐based amplification‐free system with CRISPR/CAS‐dependent enzymatic (CASCADE) assay, relies on mobile phone imaging of a catalase‐generated gas bubble signal within a microfluidic channel and does not require any external hardware optical attachments. Upon specific detection of a SARS‐CoV‐2 reverse‐transcribed DNA/RNA heteroduplex target (orf1ab) by the ribonucleoprotein complex, the transcleavage collateral activity of the Cas12a protein on a Catalase:ssDNA probe triggers the bubble signal on the system. High analytical sensitivity in signal detection without previous target amplification (down to 50 copies µL−1) is observed in spiked samples, in ≈71 min from sample input to results readout. With the aid of a smartphone vision tool, high accuracy (AUC = 1.0; CI: 0.715 – 1.00) is achieved when the CASCADE system is tested with nasopharyngeal swab samples of PCR‐positive COVID‐19 patients. The CASCADE (Cellphone‐Based Amplification‐Free System with CRISPR/CAS‐Dependent Enzymatic) assay relies on the Cas12‐mediated trans‐cleavage of a Catalase:ssDNA probe, upon recognition of a specific nucleic acid target, such as SARS‐CoV‐2 genomic RNA. This generates a gas bubble signal within a microfluidic channel which is then detected using a smartphone camera and a dedicated smartphone application, without any optical hardware attachment.

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Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy.

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