Engineered phage tail spike protein-based magnetic separation (T-MS) for rapid isolation and selective detection of viable Salmonella

Salmonella is a prevalent cause of foodborne illnesses worldwide, necessitating the development of specific and rapid detection tools. To address this issue, we engineered tail spike protein 2 (gp162) derived from Salmonella phage SFP10. A catalytically inactivated mutant lacking the N-terminal head domain (Mut) exhibited enhanced binding to Salmonella cells compared to the wild-type. Mut was fused with a silica-binding domain for immobilization on silica-coated magnetic beads, enabling TSP-based magnetic separation (T-MS). T-MS specifically captured 80 - 90% of S . Typhimurium cells in buffer, and maintained capture efficiency in various food matrices (milk, iceberg lettuce, and pork). The T-MS–ATP bioluminescence assay achieved a detection limit of 41 CFU/mL in iceberg lettuce. In addition, the method generated reliable luminescence signals in pork, chicken breast, iceberg lettuce, and milk, with recovery rates ranging from 95.19% to 107.23% of viable Salmonella cells within 30 min. Overall, T-MS coupled with ATP-bioluminescence assay offers a rapid, specific, and highly reliable method for detecting viable Salmonella cells.