A rapid detection method for Vibrio parahaemolyticus based on real-time Recombinase Polymerase Amplification (RPA) in food

A rapid detection method for Vibrio parahaemolyticus ( V. parahaemolyticus ) based on real-time Recombinase Polymerase Amplification (real-time RPA) was developed. The assay exhibited high specificity, successfully amplifying target genes within 20 min at 39 °C, with a detection limit (LOD₅₀) of 114 CFU/mL. To assess detection performance, artificially contaminated food samples—including fish sauce, salmon, and seafood rice balls—were tested. An enrichment step at 36 °C for 6 h was applied before real-time RPA detection. The method demonstrated robust performance across various food matrices, with relative limits of detection (RLOD) values below 2.5 compared to GB 4789.7, the mandatory national standard of China for V. parahaemolyticus detection. Validation was conducted in accordance with GB 4789.45, which outlines the general principles for microbiological method validation of China. Moreover, comparative testing of bulk fish samples using both real-time RPA and the GB 4789.7 standard method yielded consistent results. These findings highlight the potential of real-time RPA as a reliable and efficient alternative for V. parahaemolyticus detection, contributing to improved food safety monitoring. • Real-time RPA detects V. parahaemolyticus within 7 h with LOD₅₀ of 114 CFU/mL. • The method was validated per GB 4789.45 with relative sensitivity under 2.5. • Food matrices were selected base on GB 29921: fish sauce, salmon, and rice balls. • Field tests showed high agreement with GB 4789.7 for pathogen detection.