A Hydroxynaphthol Blue-Based Loop-Mediated Isothermal Amplification Assay for Closed-Tube Detection of the Streptomycin Resistance Gene aadA1 in Salmonella.

The aadA1 gene, which confers resistance to streptomycin, is typically located within class Ⅰ integrons. This genetic context enables its dissemination among diverse Gram-negative bacteria, such as Salmonella, and facilitates its potential transfer to humans through the food chain or into agricultural environments via manure. Hence, the detection of aadA1 genes is crucial for surveillance, understanding transmission dynamics, and informing strategies to mitigate the spread of resistant bacteria. Conventional aadA1 detection relies on time-consuming or equipment-intensive molecular assays like PCR or qPCR. In this study, we developed and optimized a closed-tube, hydroxynaphthol blue (HNB) -based loop-mediated isothermal amplification (LAMP) assay to detect Salmonella aadA1 gene and performed evaluation and validation against conventional PCR. The LAMP assay demonstrated high specificity and sensitivity, with a detection limit of 1 pg (190 copies) of genomic DNA per reaction, which is tenfold higher than that of conventional PCR. In parallel testing of 40 Salmonella DNA samples, the optimized LAMP assay achieved a detection rate of 100.0% for the aadA1 gene in streptomycin-resistant isolates, compared to 96.3% by conventional PCR. Among the streptomycin-susceptible isolates, the LAMP assay also showed a higher detection rate (38.5%) for the aadA1 gene, compared to 23.1% by conventional PCR. Consequently, the LAMP assay developed in this study for detecting the aadA1 gene offers a combination of simplicity, speed, visual readout, high specificity, and sensitivity, making it particularly suitable for rapid field detection in antimicrobial resistance surveillance programs.