On-site detection of extracellular antibiotic resistance genes by magnetic solid phase extraction-light driven amplification-lateral flow assay

Abstract

Antibiotic resistance genes (ARGs), especially extracellular ARGs (eARGs), are recognized as emerging pollutants that pose significant threats to public health and the environment, characterized by their increased transmission risk across bacterial genera and the difficulty in monitoring them. Herein, a comprehensive method for the efficient detection of eARGs was developed, integrating magnetic solid phase extraction (MSPE), light-driven (LD) recombinase polymerase amplification (RPA), and lateral flow assay (LFA). Initially, Fe₃O₄@polydopamine (Fe₃O₄@PDA) was utilized as the adsorbent in MSPE to extract eARGs from samples. The extracellular sulfonamide resistance gene sul1 was chosen as a model analyte for this study. Subsequently, the efficiency of nucleic acid amplification was significantly enhanced by 2.2 times compared to traditional water-bathed RPA, through the implementation of LD-RPA. This technique employed a portable laser flashlight as the light source and Fe₃O₄@PDA as the photosensitive medium, facilitating rapid and efficient amplification of the target gene. Following amplification, the specifically designed LFA was utilized for the detection of the amplicons. The resulting assay demonstrated a wide linear range of 10 copies/µL to 1.0 × 10⁵ copies/µL in the eluent of MSPE (R² = 0.9939), with a remarkable detection limit of 10 copies/µL eluent, corresponding to 3.3 × 10⁻² copies/µL in water samples. The entire analytical procedure costs only 35 min. To validate the method, environmental water samples were tested, and the results obtained were found to be in good agreement with those from real-time quantitative polymerase chain reaction, further substantiating the accuracy and efficacy of the integrated MSPE–LD-RPA–LFA approach.