Enhanced recombinase polymerase amplification via UvsX engineering and reaction optimization for rapid detection of Vibrio parahaemolyticus.

Recombinase polymerase amplification (RPA) is a powerful isothermal nucleic acid amplification technique, yet its efficiency is critically dependent on the catalytic efficiency of the recombinase UvsX, a key enzyme mediating homologous DNA pairing and strand exchange. To address this limitation, in this study, we developed a specific, sensitive, and robust RPA detection method by optimizing the UvsX enzyme through protein engineering and refining the RPA reaction system. By conducting comparative structural and functional analysis of UvsX orthologs from 13 Myoviridae phages, we identified critical determinants of recombinase activity within the Loop 2 domain of T4 UvsX. Furthermore, we systematically optimized the stoichiometric ratios of core enzymes and crowding agents to establish a robust RPA system. This system was subsequently integrated with lateral flow strips for point-of-need detection of highly lethal Vibrio parahaemolyticus in shrimp. Our results demonstrated that the engineered UvsXv1 variant exhibited significantly improved strand displacement activity, leading to enhanced RPA amplification efficiency and stability.