Apta-NEM-Ago: An allosteric aptamer-triggered NEMA-TtAgo fluorescence biosensor for Salmonella Enteritidis detection

Rapid and sensitive detection of Salmonella Enteritidis (S. Enteritidis) in complex matrices remains challenging because conventional methods commonly require bacterial enrichment, nucleic acid extraction, or thermal cycling. Here, we developed Allosteric aptamer-triggered NEMA-TtAgo (Apta-NEM-Ago), a modular fluorescence biosensor that couples allosteric aptamer probe (AP)-mediated whole-cell recognition with nicking enzyme-mediated amplification (NEMA) and Thermus thermophilus Argonaute (TtAgo)-mediated signal output. Binding of intact S. Enteritidis induced conformational switching of AP and exposed a template-binding domain that initiated NEMA. The resulting 5'-phosphorylated trigger strands directly served as guides for TtAgo, enabling sequence-specific cleavage of a fluorophore-quencher reporter and fluorescence recovery. Under the optimized conditions, the assay exhibited a linear response from 10° to 10³ CFU/mL and a calculated limit of detection of 0.32 CFU/mL. The recognition, amplification, and fluorescence-readout steps were completed within approximately 75 min. The assay selectively distinguished S. Enteritidis from nine non-target bacterial species and retained stable analytical performance after the prepared reaction solutions were stored at -20 °C for 14 days. Recoveries ranged from 89.6% to 106.2% in milk and from 102.8% to 110.5% in serum, and the classification of nine fecal samples agreed with culture-based analysis. By integrating direct whole-cell recognition, rapid isothermal amplification, and programmable nuclease-mediated readout, Apta-NEM-Ago provides a nucleic acid extraction-free and adaptable analytical strategy for S. Enteritidis detection in food and biological samples.