FRET-Based Single-Molecule Detection of Pathogens

Abstract The prevalence of foodborne diseases (FBDs) has been a big challenge in the food industry. Among several, Staphylococcus aureus is one of the most common foodborne pathogens, causing significant challenges to public health and food safety worldwide. While various detection strategies are currently availablefrom culture-based methods to modern biosensorsthese platforms still face notable drawbacks including long culture time and poor accuracy; there is a need for improved approaches that enhance detection efficiency. Here, we introduce a novel detection of S. aureus via a specific gene segment16SSAIII, a 16S ribosomal RNA domain containing a hypervariable region and species-specific sequence, making it the most suitable region for identification of pathogens. The detection is achieved via a change in the FRET (fluorescence resonance energy transfer) signal upon binding of the target to a custom-designed DNA platform containing multiple binding sites. The developed detection platform integrates three identical donor−acceptor FRET pairs to enable enhanced binding and improved sensitivity. This strategy enabled detection down to 1 pM without target amplification and labeling and 1 fM with increased incubation time and ionic strength. The FRET-based single-molecule detection of S. aureus that we reported here can be adapted for reliable and sensitive detection of a wide range of pathogens, ensuring food safety, and may find applications in clinical diagnostics, detection of viruses, environmental monitoring, and beyond.