Title page for ETD etd-06232010-092447


Type of Document Master's Thesis
Author Chen, Siyi
Author's Email Address csiyi1@lsu.edu
URN etd-06232010-092447
Title Detect Live Salmonella Cells in Produce by Coupling Propidium Monoazide with Loop-Mediated Isothermal Amplification (PMA-LAMP)
Degree Master of Science (M.S.)
Department Food Science
Advisory Committee
Advisor Name Title
Ge, Beilei Committee Chair
Finley, John W. Committee Member
Prinyawiwatkul, Witoon Committee Member
Keywords
  • PMA-LAMP
  • produce
  • live detection
Date of Defense 2010-05-10
Availability unrestricted
Abstract
Salmonella is a leading cause of foodborne illnesses worldwide. In recent years, an increasing number of Salmonella-related outbreaks in produce has been reported. It is therefore important that the produce industry be equipped with rapid, sensitive, specific detection methods for live Salmonella cells in produce to better ensure the produce safety. In this study, we first designed and optimized a loop-mediated isothermal amplification (LAMP) assay for Salmonella detection by targeting the invasion gene (invA). Then we incorporated a chemical reagent, propidium monoazide (PMA) into the sample preparation step to prevent LAMP amplification of dead Salmonella cells. To our knowledge, this is the first study that combined these two novel technologies for live bacterial detection. The PMA-LAMP was evaluated for false positive exclusivity, sensitivity, and quantitative capability. Finally, the PMA-LAMP assay was applied to detect live Salmonella cells in the presence of dead cells in several produce items (cantaloupe, spinach, and tomato). The invA-based PMA-LAMP could avoid detecting heat-killed dead Salmonella cells up to 7.5 105 CFU per reaction and could detect down to 3.4 - 34 live Salmonella cells in the presence of 7.5 103 heat-killed dead Salmonella cells per reaction in pure culture with good quantitative capability (r2 = 0.983). When applied to produce testing, the assay could avoid detecting heat-killed dead Salmonella cells up to 3.75 108 CFU/g and could successfully detect down to 5.5 103 - 5.5 104 CFU/g of live Salmonella cells in the presence of 3.75 106 CFU/g of heat-killed Salmonella cells with good quantitative capability (r2 = 0.993 - 0.949). The total assay time was 3 hours. When compared with PMA-PCR, the PMA-LAMP assay was 10 to 100-fold more sensitive, 2-hour shorter, and technically simpler. In conclusion, the invA-based PMA-LAMP assay developed in this study was an effective tool to specifically detect live Salmonella cells in produce with high sensitivity and quantitative capability.
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