文献类型：期刊文献

英文题名：Enhanced recombinase polymerase amplification via UvsX engineering and reaction optimization for rapid detection of Vibrio parahaemolyticus

作者：Wang, Lin[1];Li, Yiming[1];Wang, Pengbo[2];Zhang, Yibei[1,2,3];Liu, Qin[1,2,3]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Shanghai Engn Res Ctr Maricultured Anim Vaccines, Shanghai 200237, Peoples R China;[3]Lab Aquat Anim Dis MOA, Shanghai 201400, Peoples R China

年份：2025

卷号：78

期号：6

外文期刊名：LETTERS IN APPLIED MICROBIOLOGY

收录：;EI(收录号：20252618676961);WOS:【SCI-EXPANDED(收录号:WOS:001517837200001)】；

基金：Thanks to the School of Biotechnology of East China University of Science and Technology. Thanks to the Third Institute of Oceanography, Ministry of Natural Resources for the generous gift of the Vibrio strains.

语种：英文

外文关键词：recombinase polymerase amplification (RPA); recombinase UvsX; reaction optimization; Vibrio parahaemolyticus; rapid detection

摘要：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.