Development of a Novel Loop-Mediated Isothermal Amplification (lamp) Assay for the Detection of Salmonella Ser. Enteritidis from Egg Products

Abstract Salmonella ser. Enteritidis is a major public health concern worldwide. Loop-mediated isothermal amplification (LAMP) is a novel simple, easy-to-operate detection technology that amplifies DNA with high speed, efficiency, and specificity under isothermal conditions. The objective of this study was to develop a new LAMP assay for the detection of Salmonella ser. Enteritidis. The Prot 6E gene located on a virulence plasmid of Salmonella ser. Enteritidis, encoding fimbrial biosynthesis protein, was the target for detection. The primer set was designed by using Primer Explorer V4 software and evaluated for its effectiveness in detecting Salmonella ser. Enteritidis strains SE12 (PT14b), 18579 (PT4), and CDC_2010K_1441 (PT8) using isothermal master mix under an approximately 35 min reaction by Genie III instrument (OptiGene, UK). Ratio of outer and inner primers, amount of DNA template, reaction temperature and time were optimized. Inclusivity test using 114 Salmonella ser. Enteritidis strains showed 97.4% positive for prot 6E gene. For exclusivity testing, 34 non- Salmonella ser. Enteritidis Salmonella strains (27 serotypes) and 35 non- Salmonella strains (14 species) were tested and they were 100% negative. Results from the LAMP assay on 200 samples from egg products inoculated with 1–5 CFU/25 g completely matched with that from culture method and real-time PCR, and less than 1.2–12 CFU per reaction Salmonella ser. Enteritidis could be detected. This LAMP method can be of high value to the food industry due to its various advantages such as speed, specificity, sensitivity, cost- and labor-efficiency.