Real-time and visual detection of viable Salmonella in milk from remote pasture via IMS-LAMP-NALFS

Salmonella is a prevalent foodborne pathogen commonly found in dairy products. Controlling the presence of Salmonella during dairy product production is of utmost concern due to the potential for contamination at any stage of the production chain, particularly at the source. Moreover, the unique location of pastures and limited testing resources further highlight the necessity of establishing a rapid and precise detection method to ensure milk quality control and enhance overall farm safety. To address these issues, in this study, we constructed a method as well as a portable toolbox for rapid on-site detection of Salmonella. The method involves immunomagnetic separation (IMS) using streptavidin-coupled magnetic nanoparticles and biotinylated antibodies. The reaction conditions for magnetic capture and enrichment of Salmonella were optimized to efficiently remove matrix interference from milk and enhance detection sensitivity. Additionally, an improved propidium monoazide (PMAxx) treatment was employed to eliminate false positive results caused by dead bacteria. This treatment was combined with loop-mediated isothermal amplification (LAMP) and nucleic acid lateral flow strip (NALFS) (LAMP-NALFS) for on-site detection of viable bacteria in milk. The resulting IMS-LAMP-NALFA assay achieved rapid and sensitive detection of viable Salmonella, with a detection sensitivity of 1 x 102 CFU/mL in milk samples, completed within 90 min under optimal conditions. Based on this achievement, a portable toolbox was designed for easy transportation and on-site testing in pastures. It is equally applicable for rapid detection of viable Salmonella contamination in food products in resource-limited areas such as pastures, where the observed detection rate of Salmonella in unpasteurized raw milk was 7.93 % (28 out of 353 samples tested). Consequently, the IMS-LAMP-NALFA method and the associated portable toolbox represent highly effective means of enhancing the detection of viable Salmonella in dairy products.