Visual detection for environmental antibiotic resistance genes by recombinase polymerase amplification combined with lateral flow dipstick

Abstract The emergence of the antibiotic resistance genes(ARGs) has posed a significant challenge in controlling the spread of multidrug-resistant bacteria both in clinic and environment.To establish the purpose of detecting the environmental ARGs in room temperature and achieving visualization, this study developed a rapid detection by combining recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) targeting crucial ARGs including blaNDM, blaKPC, mcr, tetX1 and tetX2. Various primers and nfo probes were designed and LFD-RPA method was established, followed by the sensitivity, specificity and repeatability tests.Sensitivity was evaluated with ten-fold serial dilutions of plasmid standards, specificity was tested against environmental isolates lacking target genes, and reproducibility was assessed in ten replicates. Results showed that the limits of detection ranged from 1.061×10¹ to 1.803×10²copies/mL,with tetX1 demonstrating the highest sensitivity at 1.061×10¹ copies/mL .The LFD-RPA system showed 100% specificity without cross-reactivity, and exhibited great repeatability. Thus, the LFD-RPA platform provided a highly sensitive, specific and one-site detection for real-time environmental monitoring of key ARGs.