Development of an RT-LAMP Assay for Detecting (M) in Species: Enhancing AMR Surveillance Within the One Health Sectors.

The increasing prevalence of antimicrobial-resistant (AMR) bacteria in humans, animals, and the environment underscores the necessity for a rapid, sensitive, and specific method to identify resistance genes. : This study aims to develop a reliable detection tool for identifying the tetracycline-resistant gene (M) in species using a real-time loop-mediated isothermal amplification (RT-LAMP) assay. Real-time visualization through a turbidimeter enabled precise estimation of time-to-positivity for gene detection. : Six primers were designed using PrimerExplorer v.5, and the assay was optimized across different temperatures and incubation times. Validation was conducted by testing 52 (M)-positive clinical enterococci isolates and spiking urine samples from a healthy volunteer and a cow with (M)-positive species. The (M) gene was detected as early as 33 min, with optimal amplification occurring within 60 min at 60 °C. The assay demonstrated 100% specificity with the established primers. The sigmoidal graphs were corroborated with visual confirmation methods, including a green color change (visible to the naked eye), green fluorescence (under UV light), and a 200 bp PCR product observed via agarose gel electrophoresis. Notably, the (M) RT-LAMP assay exhibited a detection limit of 0.001 pg/μL, significantly surpassing conventional PCR, which had a detection limit of 0.1 pg/μL. : This rapid, cost-effective, highly sensitive, and specific (M) RT-LAMP assay holds significant promise as a surveillance tool for antimicrobial resistance monitoring within a One Health framework, particularly in low-resource countries.