Sensitive On-Site Detection of Antibiotic Resistance Genes in Aquatic Products by aPCR-LFA Leveraging AuNPs for Amplification Specificity and Hybrid Probes for Structural Control

The transmission of antibiotic resistance genes (ARGs) through the food chain constitutes a serious public health risk by potentially leading to drug-resistant infections in humans. Rapid on-site detection of ARGs is therefore urgently needed. In this study, we developed a lateral flow assay (LFA) combined with asymmetric PCR (aPCR) for rapid and visual detection of four major ARGs: <i>sul1</i>, <i>bla</i>_NDM-1, <i>vanA</i>, and <i>vgaB</i>. The aPCR method efficiently generated single-stranded DNA amplicons without chemical modification, making it ideal for LFA detection. To improve assay performance, gold nanoparticles (AuNPs) were introduced to suppress nonspecific amplification during aPCR. Hybrid nucleic acid probes were designed to prevent single-stranded DNA (ssDNA) self-folding and facilitate the formation of a single-stranded tail structure, enabling dual recognition between the AuNP-labeled probe and the test zone capture probe. The optimized aPCR-LFA assay detected as low as 10 copies/μL of each ARG within 40 min and showed a linear quantitative range from 10¹ to 10⁵ copies/μL. No cross-reactivity was observed among the four ARGs, confirming high specificity. The method was successfully applied to shrimp and river snail samples, producing results consistent with quantitative real-time PCR (qPCR) analysis. These findings demonstrate that aPCR-LFA is a sensitive, specific, and reliable platform for on-site monitoring of ARGs in aquatic products.