DNA Fragment Fusion-Triggered Label-Free Detection of Listeria monocytogenes Subtypes via Fusion RAA and Hybrid Split G-Quadruplex

Bacterial subtype detection using multiplex amplification with multiple distinguishable labels is complex and costly, whereas recombinant DNA technology via fragment fusion provides a simpler, cheaper alternative. Here, we propose a label-free method for Listeria monocytogenes subtype detection based on a hybrid split G-quadruplex (G4) and fusion recombinase-aided amplification (RAA)-cascaded CRISPR/Cas13a (HGFRC) platform. Fusion RAA co-amplified species-specific and clonal complex (CC)-specific targets into 535 bp fusion products, which activated T7 transcription and Cas13a trans-cleavage activity. Activated Cas13a cleaved the RNA linker, disrupting G4 assembly and reducing fluorescence to generate a signal-off response. This design eliminated labeled probes, enabling single-signal detection. The assay achieved a sensitivity of 1.0 × 101 CFU/mL within 45 min, with good specificity and sample suitability. Overall, this study provides a label-free, sensitive, and specific method for detecting L. monocytogenes CC5, offering new insights for bacterial subtype biosensing.