Rapid Detection of Listeria Monocytogenes in Salad by Polymerase Chain Reaction
Type of DegreeThesis
DepartmentNutrition and Food Science
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The development of rapid detection methods for the testing of ready-to-eat products is an area of high importance, not only for the food industry but also for consumers. The USDA standard for zero tolerance of Listeria monocytogenes in ready-to-eat products causes massive recalls that can be fatal to small food industries. In order to combat this growing problem, it is important for food industry to find the presence of L. monocytogenes in their products as soon as possible to avoid these potentially hazardous impacts. The development of the polymerase chain reaction (PCR) technique has been a huge leap forward in the development of rapid, specific and sensitive methods for the detection of food borne pathogens. The principle behind the PCR method is the amplification of a gene sequence that is specific to the targeted pathogen. After amplification, the sample can be analyzed through agarose gel electrophoresis. In this study, a set of primers were developed to amplify 400 base pairs of DNA, a segment of the internalin A gene. This protein is link to the pathogenesis of L. monocytogenes, which causes listeriosis in humans. Therefore, this gene is only found in the pathogenic strain of L. monocytogenes. The specificity of the primers was tested against 34 different bacteria and only the pathogenic L. monocytogenes isolates showed positive results on the agarose gel. Five different isolates of L. monocytogenes were tested to determine the detection sensitivity of PCR, and the results showed that it was able to detect as low as 298 cfu. Six different enrichment broths of modified Penn State University (mPSU), Listeria enrichment broth (LEB), TrypticÒ soy broth plus 0.6% yeast extract (TSBYE), half Fraser’s broth, University of Vermont medium (UVM), and half TrypticÒ soy broth plus 0.6% yeast extract (1/2 TSBYE) were tested to determine the growth rate of the bacterium in 6 hr. The TSBYE, LEB, mPSU, and UVM were chosen and tested to determine the recovery level of L. monocytogenes cells from inoculated salad. From the results, TSBYE, mPSU and LEB were chosen to represent both selective and non-selective enrichment media for L. monocytogenes enrichment on inoculated salad in PCR detection. In this study, 25 grams of ready-to-eat salad were inoculated with L. monocytogenes at 200 cfu/g for use. After blended with a stomacher, the sample was filtered through glass wool to remove large food partials, and then the filtrate was centrifuged to concentrate the bacteria and enriched in selected broths. Following 6 hr enrichment, the samples were centrifuged again to concentrate bacteria for PCR amplification. Samples were also spread plated on modified Oxford medium for actual bacterial counts. The final bacterial concentrations of TSBYE, LEB, and mPSU were 4.0 x 104, 8.2 x 103, 6.8 x 102 cfu/g, respectively. Positive PCR results were shown only on the LEB sample. This study showed that by using the internalin A based primers, filtration, centrifugation, and a 6 hour enrichment process; the PCR technique can detect L. monocytogenes at 103 cfu/g of ready-to-eat salad.