CRISPR/Cas12a coupled with loop-mediated isothermal amplification and lateral flow assay for SARS-CoV-2 detection

Abstract

Point-of-care testing (POCT) is rapid, exhibits highly sensitive performance, can facilitate home self-testing and avoids cross-contamination. Herein, we developed a biosensor that combines Si–OH magnetic bead (MB)-based fast RNA extraction, reverse transcription-loop-mediated isothermal amplification (RT-LAMP), CRISPR-Cas12a, and lateral flow assay (LFA) for rapid detection of SARS-CoV-2 RNA within 1.5 h. In the presence of the SARS-CoV-2 LAMP amplicon, the trans-cleavage activity of Cas12a was activated to cleave the probe, separating streptavidin from the AuNPs-digoxin (Dig) antibody, resulting in the inability of the test line to capture the AuNPs-Dig antibody. The method can distinguish SARS-CoV-2 from other RNA viruses, with a limit-of-detection (LOD) of 6.2 × 10² copies per mL. Therefore, LAMP-CRISPR-LFA has high specificity and sensitivity and is convenient to develop into commercial assay kits, which could have a broad prospect for practical application.