Cas12aFDet: A CRISPR/Cas12a-based fluorescence platform for sensitive and specific detection of Listeria monocytogenes serotype 4c

The CRISPR/Cas12a system has displayed remarkable potential in the development of new methods for nucleic acid detection owing to the trans-cleavage activity of Cas12a. Despite the tremendous development in recent years, existing CRISPR/Cas12a-based methods have several limitations such as the time-consuming process, which takes up to 2 h, and the risk of aerosol contamination during DNA amplicon transfer. Herein, we propose a CRISPR/Cas12a-based fluorescence detection platform named Cas12aFDet for rapid nucleic acid detection that overcomes these limitations. By integrating PCR or recombinase-aided amplification (RAA) methods with Cas12a-mediated cleavage in a sealed reaction tube, Cas12aFDet-based detection of amplified products could be accomplished within 15 min, while avoiding amplicon contamination. The detection limits of PCR-based Cas12aFDet and RAA-based Cas12aFDet were determined to be 3.37 x 10(1) cfu/mL and 1.35 x 10(2) cfu/mL of Listeria monocytogenes serotype 4c in pure culture, respectively. Most importantly, RAA-based Cas12aFDet exhibited 0.64 aM sensitivity for DNA detection, and showed high specificity for detection of other serotypes of Listeria and non-Listeria strains. Furthermore, the feasibility of the RAA-based Cas12aFDet method was evaluated in spiked and natural samples, enabling the quantitative detection of 1.35 x 108e1.35 x 103 cfu/g fresh grass carp of the target L. monocytogenes serotype 4c, and the results obtained for 22 natural aquatic samples were highly consistent with those of the culture-based serotyping method. The established Cas12aFDet platform is expected to provide a new paradigm for the sensitive and specific detection of pathogens in food safety and clinical diagnosis. (c) 2021 Elsevier B.V. All rights reserved.

Automated summary

The study introduces a CRISPR/Cas12a-based fluorescence detection platform called Cas12aFDet for rapid nucleic acid detection, which overcomes limitations of existing methods such as time consumption and contamination risk. The platform demonstrates high sensitivity and specificity for detecting Listeria strains in both pure culture and natural samples.