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.
| Identifer | oai:union.ndltd.org:LSU/oai:etd.lsu.edu:etd-06232010-092447 |
| Date | 23 June 2010 |
| Creators | Chen, Siyi |
| Contributors | Prinyawiwatkul, Witoon, Ge, Beilei, Finley, John W. |
| Publisher | LSU |
| Source Sets | Louisiana State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lsu.edu/docs/available/etd-06232010-092447/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached herein a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to LSU or its agents the non-exclusive license to archive and make accessible, under the conditions specified below and in appropriate University policies, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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