A Combination of Novel Nucleic Acid Cross-Linking Dye and Recombinase-Aided Amplification for the Rapid Detection of Viable Salmonella in Milk

Salmonella, as an important foodborne pathogen, can cause various diseases, such as severe enteritis. In recent years, various types of nucleicacid-intercalating dyes have been utilized to detect viable Salmonella. However, in principle, the performance of existing nucleic acid dyes is limited because they depend on the integrity of cell membrane. Herein, based on the metabolic activity of bacteria, a novel DNA dye called thiazole orange monoazide (TOMA) was introduced to block the DNA from dead bacteria. Recombinase-aided amplification (RAA) was then performed to detect viable Salmonella in samples. In this study, the permeability of TOMA to the cell membrane of Salmonella was evaluated via confocal laser scanning microscopy and fluorescence emission spectrometry. The limit of detection (LOD) of the TOMA–RAA method was 2.0 × 10(4) CFU/mL in pure culture. The feasibility of the TOMA–RAA method in detecting Salmonella was assessed in spiked milk. The LOD for Salmonella was 3.5 × 10(2) CFU/mL after 3 h of enrichment and 3.5 × 10(0) CFU/mL after 5 h of enrichment. The proposed TOMA–RAA assay has great potential to be applied to accurately detect and monitor foodborne pathogens in milk and its byproducts.