Simultaneous Detection of Vibrio parahaemolyticus and Tetracycline Resistance Genes Using Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Technology

By integrating the technical advantages of recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD), a duplex RPA-LFD method for the simultaneous and rapid visual detection of Vibrio parahaemolyticus and tetracycline resistance genes in foods was developed. Multiple sets of primers and probes targeting the species-specific gene vps2310 and the tetracycline resistance gene tetA were designed and synthesized. The method was comprehensively evaluated through analytical means, such as specificity analysis, sensitivity analysis and artificial contamination experiment. The results showed that the primer-probe set selected could simultaneously detect the two targets with no cross-reactivity with non-target bacteria, demonstrating excellent specificity. The optimal reaction temperature for RPA was 40 ℃, the optimal primer ratio of vps2310 to tetA was 2:1, and the optimal reaction time for RPA was 25 min. The limit of detection (LOD) determined using pure cultures was 2.04 × 10² CFU/mL. Furthermore, the LOD of the established method was 2.60 × 10² CFU/mL for artificially contaminated samples after enrichment for 2 h. This method has the advantages of simple operation and low equipment requirements, filling the gap in the simultaneous detection of V. parahaemolyticus and resistance genes. The entire assay can be completed within 30 min, which is potentially helpful for early identification of high-risk drug-resistant bacteria.