|HU, LIJUN - Food And Drug Administration(FDA)|
|MA, LI - Oklahoma State University|
|ZHENG, SHIMIN - Northeast Agricultural University|
|HAMMACK, THOMAS - Food And Drug Administration(FDA)|
|BROWN, ERIC - Food And Drug Administration(FDA)|
|ZHANG, GUODONG - Food And Drug Administration(FDA)|
Submitted to: Food Control
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/5/2018
Publication Date: 2/2/2018
Citation: Hu, L., Ma, L., Zheng, S., He, X., Hammack, T.S., Brown, E.W., Zhang, G. 2018. Development of a novel loop-mediated isothermal amplification (LAMP) assay for the detection of Salmonella ser. Enteritidis from egg products. Food Control. 88:190-197. https://doi.org/10.1016/j.foodcont.2018.01.006.
Interpretive Summary: Salmonella is estimated to cause more than 1.2 million illnesses each year in the United States, with more than 23,000 hospitalizations and 450 deaths. Salmonella ser. Enteritidis is considered the most frequent Salmonella serotype causing salmonellosis outbreaks associated with poultry, eggs, and egg products, due to its unique ability to contaminate the egg contents through vertical transmission. This manuscript evaluated a new technology called Loop-mediated isothermal amplification (LAMP) for detection of Salmonella in egg products. The assay developed in this study was proven to be as effective as FDA’s BAM Salmonella culture and real-time PCR methods, but was much simpler and faster, making it an ideal complementary method to existing approaches for quick and accurate detection of Salmonella ser Enteritidis in foods.
Technical 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 Prot6E 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 35min 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 prot6E 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 1e5 CFU/25 g completely matched with that from culture method and real-time PCR, and less than 1.2e12 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.