Simultaneous detection of Vibrio parahemolyticus and antimicrobial resistance genes using immonomagnetic separation combined with RPA-microfluidic method in seafood

Rapid, accurate, and simultaneous screening of V. parahaemolyticus and its antimicrobial resistance genes (ARGs) is significant for mariculture monitoring and food safety. Thus, we developed an integrated RPA-microfluidic method to simultaneously detect V. parahaemolyticus and its ARGs floR, sul1 and qnrA. First, we used the optimized immunomagnetic separation (IMS) to enrich the target bacteria, and the capture efficiency of V. parahaemolyticus (104 CFU/mL) can reach 91.87% ± 1.8% after incubation for 45 min and IMS time of 4 min. Then using boiling method (∼5 min) to extract DNA and the RPA-microfluidic method was performed at 39 °C for 20 min. The results demonstrated that the limit of detection reached 102 copies/μL for all target genes with a specificity of 100%. In conclusion, our IMS-RPA-microfluidic method demonstrated significant advantages, including time efficiency, user-friendly operation, and high sensitivity. This method holds great potential for the on-site monitoring of foodborne pathogenic bacteria and ARGs.