Gelatin assisted RPA-CRISPR/Cas12a tandem assay for rapid detection of Listeria monocytogenes

Abstract Methods integrating isothermal amplification with the CRISPR/Cas12a system have gained significant attention due to their sensitivity and specificity. Herein, a gelatin-assisted recombinase polymerase amplification (RPA)-CRISPR/Cas12a tandem (GARC) assay was developed to detect Listeria monocytogenes . The GARC assay design exploited temperature-responsive gelatin to physically encapsulate CRISPR/Cas12a reagents, maintaining them in a gelled state during RPA incubation at 37 °C. This spatial separation prevents premature interaction between RPA and CRISPR components, while the gelatin at an appropriate concentration enhances fluorescence signal intensity, facilitating visual readout. The optimized GARC assay achieved a limit of detection (LOD) of 10 ag/μL for the hlyA gene, representing 10 4 and 10 7 fold improvements over paraffin-based one-pot RPA-CRISPR (100 fg/μL) and conventional PCR methods (100 pg/μL), respectively. Notably, the assay detected 8 CFU/mL of L. monocytogenes and 1.03 × 10 −5 ng/μL of genomic DNA, with favorable sensitivity (5/5) and specificity (16/16). Practical applicability was demonstrated through the successful detection of L. monocytogenes in salmon samples after 7-h enrichment at 20 CFU/25 g. This work introduces a versatile, visually interpretable platform with potential for food safety testing.