Development and evaluation of molecular-based assays for detecting Salmonella serovars in various food commodities

As a leading cause of foodborne illnesses and outbreaks, Salmonella poses a major public health risk in the United States and worldwide. Various food commodities including meat and poultry, eggs, and fresh produce can serve as the transmission vehicles for Salmonella infections. To better ensure the safety of these products and protect public health, rapid, accurate, and reliable detection methods for Salmonella are needed. Molecular-based methods like loop-mediated isothermal amplification (LAMP), have gained wide applications in Salmonella detection, owning to their rapidity, specificity, and sensitivity. However, there is a paucity of data on the robustness of these assays. And very recently, bioluminescence assay in real-time (BART) was used as a new and effective platform to detect LAMP products, and this combination has not been evaluated before. This dissertation research evaluated the robustness of two LAMP assays in comparison with PCR, examined the application of LAMP assays in detecting Salmonella specifically in food items, and developed a novel LAMP-BART assay for Salmonella detection. The LAMP assays achieved robust detection of Salmonella under abusive preparation and running temperatures, also demonstrated greater tolerance than PCR to various inhibitors. They achieved 100% accuracy among 185 strains. The limits of detection of LAMP for Salmonella strains belonging to ten serovars were 1 to 10 cells per reaction in pure culture, 100-fold more sensitive than PCR. In spiked egg homogenates, it could detect Salmonella serovars Enteritidis and Typhimurium down to 10^4 CFU/25 ml egg homogenates directly and 1 CFU/25 ml with 8 h enrichment. In spiked produce (cantaloupe, jalapeno pepper, tomatoes, sprouts, and lettuces), the detection limits ranged from 10^4 to 10^6 CFU/25 g produce, which were comparable to qPCR. Coupled with 6 to 8 h of enrichment, LAMP consistently detected in produce samples spiked with very low levels of Salmonella cells, with the exception of sprouts. Based on these evaluations and further development, LAMP demonstrated to be a rapid and robust alternative to PCR-based assays for Salmonella detection and could be adopted by food industries and regulatory agencies in routine product testing for Salmonella to improve product safety and protect public health