Salmonella Typhimurium (S. Typhimurium) is an important foodborne pathogen, its harm is mainly reflected in high pathogenicity, clinical manifestations, and drug resistance. The detection technology for this pathogen is gradually shifting from traditional methods to rapid and highly sensitive molecular biological methods. In order to solve the sensitivity and specificity of Salmonella detection, we built a fluorescent biosensor with an antibody-target-aptamer sandwich structure based on immune recognition and aptamer technology for quantitative detection of S. Typhimurium. By integrating the heterogeneous recognition mode composed of antibody and aptamer with isothermal hybridization chain reaction (HCR), this approach enables highly specific detection of Salmonella while significantly enhancing detection sensitivity through signal amplification. In the HCR hairpin structure, site-specific labeling of the fluorophore and quencher enables fluorescence signal generation via fluorescence resonance energy transfer (FRET), thereby achieving efficient optical signal output. The results show that the proposed enzyme-free heterogeneous recognition mode sensor has a detection limit of 9 CFU/mL and a 10-10⁴ CFU/mL detection range under optimized conditions. It also has good detection performance in milk and drinking water, which are common sources of S. Typhimurium contamination.

Foodborne pathogens, a major cause of foodborne disease outbreaks, are a great threat to human life and health. In this context, rapid and accurate detection of foodborne pathogens is of great interest. In recent years, electrochemical biosensors as a fast, sensitive and simple detection method have been widely studied, and most studies focus on the development and immobilization of recognition elements and the application of electrochemical biosensors in sample detection. Phages, bacterial viruses specifically infect their hosts, can be used as an important recognition element in electrochemical biosensor for the detection of foodborne pathogens. Compared with enzyme, antibody, nucleic acid and other recognition elements, phages are easier to obtain, more tolerant to the environment, and more stable. Simultaneous phage-based electrochemical techniques with impedance and amperometric probes have gained increasing attention and been applied due to their high sensitivity. In this paper, the phage immobilization methods and the current status of the application of phages in electrochemical biosensors are described, and future trends are also discussed.

Salmonella pullorum phage Pu29 was comprehensively analyzed for its biological and genomic characteristics. A magnetic separation and enrichment technique for Salmonella was established using the phage Pu29 as a recognition element. The phage belonged to the genus Roufvirus and had an icosahedron head and an irreducible long tail. Pu29 had a wide host spectrum with an adsorption rate of 88.67% on host cells in 15 min, a latent period of 30 min, a rise period of 180 min, and a burst size of 115.74 PFU/cell. Meanwhile, Pu29 had good heat resistance (30-60 ℃) and pH tolerance (pH 4-11). Its genome was composed of 45715 bp (GC content 46.08%) and 81 open reading frames (ORFs), including 18 ORFs with known functions that did not carry genes encoding toxicity or resistance factors. PhagePu29-MBs were prepared as a probe by coupling the phage with carboxylated nano-magnetic beads (MBs) through amide reaction. When 25 μg of the probe was incubated with Salmonella at 37 ℃ for 20 min, the highest capture rate of Salmonella of 83.93% and the lowest captured bacterial concentration of 45 CFU/mL were obtained. Transmission electron microscopy (TEM) was used to observe that PhagePu29-MBs could specifically capture Salmonella. In spiked samples, the highest capture rate of Salmonella separated and enriched by the probe reached 92.92%. The separation and enrichment process took approximately 30 min. Therefore, this study established a fast and highly specific magnetic separation method for Salmonella based on phage Pu29, which may lay the foundation for the development of a phage-based method for the rapid separation and enrichment of foodborne pathogens in food samples.