A Novel Approach Using LuxSit-i Enhanced Toehold Switches for the Rapid Detection of Vibrio parahaemolyticus

<i>Vibrio parahaemolyticus</i> (<i>V. parahaemolyticus</i>) is a significant concern, as it can cause severe infections and hemolytic trauma. Given its prevalence in seawater and coastal seafood, it poses a substantial risk as a foodborne pathogen. Biosensor-based detection technology has been continuously evolving, and toehold switches have emerged as a promising area within it, especially in the detection of RNA viruses. Here, we have developed a cell-free toehold switch sensor for <i>V. parahaemolyticus</i> detection. Traditional toehold switch detection methods usually use green fluorescent protein (GFP) or enzyme LacZ as the output signal, with an incubation time as long as 2 h, and are also mainly applied to the detection of RNA viruses. In this study, we introduced a novel, artificially designed luciferase (LuxSit-i) as an output signal and constructed toehold switches with two different output signals (sfGFP, LuxSit-i), aimed at reducing the incubation time of toehold switches. Moreover, to further improve the detection process, we separately utilize recombinase polymerase amplification (RPA) and nucleic acid sequence-based amplification (NASBA) to amplify dead and live bacterial suspensions for detection and attempt to distinguish between dead and live bacteria. This study provided a convenient, rapid, and accurate method for the on-site detection of <i>V. parahaemolyticus</i>, especially beneficial for resource-limited settings. By eliminating the requirement for specialized facilities and personnel, this system has the potential to be a valuable tool in improving public health responses, especially in developing regions.