Listeria monocytogenes detection assay via aptamer-functionalized magnetic bead enrichment coupled with RPA-CRISPR/Cas12a lateral flow strips

Abstract

Conventional detection methods for Listeria monocytogenes suffer from limitations including lengthy procedures, equipment dependency, and operational complexity, making them unsuitable for rapid on-site detection. Therefore, this study developed an LM-RPA-Cas12a-LFA detection method. Target bacteria were enriched using aptamer magnetic beads, followed by RPA amplification of a 196 bp fragment of the hly gene to activate the Cas12a system, with results displayed on lateral flow strips. The method including bacterial enrichment, DNA extraction, LM-RPA-Cas12a-LFA procedure required 2 h with detection limit of 1 × 10−10 ng/µL and 1.35 CFU/mL in complex food matrices, demonstrating excellent reproducibility and stability. Detection of 16 real food samples showed complete concordance with qPCR validation, accurately identifying 4 positive and 12 negative samples. This method provides a reliable, rapid, and sensitive tool for on-site qualitative detection of Listeria monocytogenes.