Visual Detection for Environmental Antimicrobial Resistance Genes by Recombinase Polymerase Amplification Combined...

The emergence of antimicrobial resistance genes (ARGs) has posed a significant challenge in controlling the spread of multidrug-resistant bacteria both in the clinic and in the environment. Traditional monitoring techniques, however, rely on laboratory instruments. This study aimed to develop and validate a room-temperature and instrument-free assay that visually reports the presence of five representative ARGs: bla_NDM, bla_KPC, mcr-1, tetX1, and tetX2. To achieve this purpose, we developed a rapid detection by combining recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) system. Various primer and probe sets were designed, and an LFD-RPA method was established, followed by the sensitivity, specificity, and repeatability tests. Sensitivity was evaluated using ten-fold serial dilutions of DNA templates, 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 × 10² copies/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 on-site detection method for real-time environmental monitoring of key ARGs.