Allosteric transcription factor (aTF)-based in vitro biosensors constitute a class of detection tools formed by the functional coupling of the ligand-binding domain of aTFs with a reporter system. Owing to advantages such as high specificity and sensitivity, these biosensors have emerged as a research hotspot in the field of modern food contaminant monitoring. Our work centers on the core aspect of engineering design and systematically elaborates on the modular design strategies for aTF-based in vitro biosensors, with a focus on the design principles of the molecular recognition system, signal amplification strategies, signal output systems, and sensing systems. Furthermore, the article summarizes the advances in the application of aTF biosensors for detecting various typical food contaminants and analyzes their performance advantages. Finally, in light of existing technical limitations, it prospectively discusses future directions for enhancing specificity, improving stability, and promoting commercial applications, aiming to provide a theoretical reference and application guidance for transitioning this technology from laboratory platforms to on-site real-time monitoring.

Accurately detecting viable foodborne pathogenic bacteria is essential for food safety risk assessments and public health interventions. Traditional plate counting is time-consuming and operationally cumbersome. Immunological assays are unable to distinguish viable from dead cells, whereas conventional nucleic acid amplification is often affected by residual DNA originating from dead bacteria. These limitations render conventional approaches inadequate for rapid and precise field detection. Functional nucleic acids (FNAs) offer a promising alternative for viability detection because of their high sensitivity, specificity, target diversity, and programmable integrability. This review provides a systematic overview of molecular recognition strategies and FNA-based detection technologies for identifying viable foodborne microorganisms. We categorize the biomarkers targeted by FNAs into nucleic acids, surface structures, and metabolic activities. Building on this categorization, we examine the core principles and technological evolution of primers, aptamers, DNAzymes, guide nucleic acids, and oligonucleotide probes in viability discrimination. We then outline the practical applications of these technologies across the food supply chain and discuss the remaining challenges and future directions in the field. Ultimately, this work provides a theoretical reference and practical guidance for ensuring food safety and advancing precise microbial risk management.