Rapid Visual CRISPR Assay: A Naked-Eye Colorimetric Detection Method for Nucleic Acids Based on CRISPR/Cas12a and a...

Rapid diagnosis based on naked-eye colorimetric detection remains challenging, but it could build new capacities for molecular point-of-care testing (POCT). In this study, we evaluated the performance of 16 types of single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporters for use with clusters of regularly spaced short palindrome repeats (CRISPR)/Cas12a-based visual colorimetric assays. Among them, nine ssDNA-FQ reporters were found to be suitable for direct visual colorimetric detection, with especially very strong performance using ROX-labeled reporters. We optimized the reaction concentrations of these ssDNA-FQ reporters for a naked-eye read-out of assay results (no transducing component required for visualization). In particular, we developed a convolutional neural network algorithm to standardize and automate the analytical colorimetric assessment of images and integrated this into the MagicEye mobile phone software. A field-deployable assay platform named RApid VIsual CRISPR (RAVI-CRISPR) based on a ROX-labeled reporter with isothermal amplification and CRISPR/Cas12a targeting was established. We deployed RAVI-CRISPR in a single tube toward an instrument-less colorimetric POCT format that required only a portable rechargeable hand warmer for incubation. The RAVI-CRISPR was successfully used for the high-sensitivity detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and African swine fever virus (ASFV). Our study demonstrates this RAVI-CRISPR/MagicEye system to be suitable for distinguishing different pathogenic nucleic acid targets with high specificity and sensitivity as the simplest-to-date platform for rapid pen- or bed-side testing.