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Rapid visual CRISPR assay: a naked-eye colorimetric detection method for nucleic acids based on CRISPR/Cas12a and convolutional neural network

By Shengsong Xie, Dagang Tao, Yuhua Fu, Bingrong Xu, You Tang, Steinaa Lucilla, Hemmink Hanneke, Wenya Pan, Xin Huang, Changzhi Zhao, Jinxue Ruan, Liangliang Fu, Xiongwei Nie, Yi Zhang, Jianlin Han, Yunlong Ma, Xinyun Li, Xiaolei Liu, Shuhong Zhao

Posted 20 Jul 2021
bioRxiv DOI: 10.1101/2021.07.17.452802

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 CRISPR/Cas12a based visual colorimetric assays. Among them, 9 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 naked-eye read-out of assay results (no transducing component required for visualization). Subsequently, we developed a convolutional neural network algorithm standardize and to 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 towards an instrument-less colorimetric POCT format that requires only a portable rechargeable hand warmer for incubation. The RAVI-CRISPR was successfully used for the single-copy detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and African swine fever virus (ASFV). Our study demonstrates this novel RAVI-CRISPR system for distinguishing different pathogenic nucleic acid targets with high specificity and sensitivity as the simplest-to-date platform for rapid pen-side testing.

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