SARS-CoV-2 RNA Detection by a Cellphone-Based Amplification-Free System with CRISPR/CAS-Dependent Enzymatic (CASCADE) Assay.

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.