Development of a visual detection method for Salmonella based on loop-mediated isothermal amplification using pyrophosphatase.

ABSTRACT

Salmonella is one of the most widely distributed and harmful food-borne pathogens; thus, the rapid detection of viable Salmonella is important for ensuring food safety. In this study, a rapid visual strategy based on loop-mediated isothermal amplification (LAMP) with the addition of thermal inorganic pyrophosphatase and linked with an ammonium molybdate chromogenic buffer was established to detect Salmonella. Specific primers were designed based on the phoP gene of Salmonella spp. The pyrophosphatase concentration, LAMP time, addition of ammonium molybdate chromogenic buffer, and color reaction time were optimized. Based on the optimal conditions, the sensitivity and specificity of the method were examined. In addition, the ability to detect actual samples was verified using apple juice containing Salmonella. LAMP was performed at 65°C for 45 min in the presence of thermal inorganic pyrophosphatase at a final concentration of 4 U ml-1, and 20 µl of the LAMP product was reacted with 50 µl of phosphate chromogenic buffer at 25°C for 15 min. According to our results, the limit of detection of the LAMP assay for viable Salmonella was 1.83 × 102 CFU per reaction, and nonspecific amplification was not observed. The detection rates of Salmonella Typhimurium with different concentrations in apple juice were 89.11%-94.80%, which verifies that the visual detection strategy is suitable for actual sample detection.