期刊
ACS SYNTHETIC BIOLOGY
卷 11, 期 1, 页码 383-396出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.1c00474
关键词
nucleic acid detection; CRISPR-Cas12a assay; convolutional neural network; MagicEye software
资金
- NSFC [31941008]
- Natural Science Foundation of China [32072685]
- Fund of Modern Industrial Technology System of Pig [CARS-35]
- CGIAR Research Program on Livestock
- CGIAR Consortium
In this study, the performance of 16 types of single-stranded DNA-fluorophore-quencher reporters for colorimetric detection in CRISPR/Cas12a-based assays was evaluated. Nine of them were found suitable for direct visual colorimetric detection, with strong performance using ROX-labeled reporters. An algorithm and mobile software were developed to standardize and automate colorimetric assessment of images, leading to the establishment of a field-deployable assay platform named RAVI-CRISPR.
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.
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