Date of Award:

2003

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Nutrition, Dietetics, and Food Sciences

Department name when degree awarded

Nutrition and Food Sciences

Advisor/Chair:

Bart C. Weimer

Abstract

Listeria monocytogenes is a foodbome pathogen that can cause severe illness and even death. It is found in dairy and meat products. The focus is on rapid detection since conventional methods are time consuming (4-5 days). Pre-enrichment steps, as part of those methods, are time consuming. Our objective was to develop a detection system without a pre-enrichment step, giving a final result within 2 to 4 h.

In the concept of "the need for speed," a detection system with an antibody-based capture technique, followed by polymerase chain reaction (PCR), was developed. Glass beads coated with a Listeria polyclonal antibody were added to the food sample. After a static incubation/capturing step, beads-cell complexes were separated from the food, and boiled to lyse the cells and release the DNA. In a final PCR/electrophoresis step the DNA samples were analyzed.

The use of a flow-based capturing system (ImmunoFlow) was also investigated. Using a bead-antibody complex in this ImmunoFlow setup has several advantages, including the possibility of concentrating the microorganisms out of large food samples (with flow through setup), the exclusion of a pre-enrichment step, and the potential for automation.

Besides buffer solution (Tris), different kinds of milk, e.g., pasteurized, Ultra High Temperature (UHT), and raw milk, were also investigated. The detection limit in buffer solution was 1 x 106 CFU/ml no matter if the ImmunoFlow system or the static incubation was used. For the different pasteurized milk samples, the detection limit varied between 1 x 107 and 1 x 108 cells/ml in the static procedure. For UHT and raw milk, however, capturing of Listeria monocytogenes cells was not possible in the static or the ImmunoFlow setup.

In conclusion, we developed a rapid and specific detection system for Listeria monocytogenes at high concentration in pasteurized milk using a static capturing procedure. The total test time for this detection system is less than 4 h, which is much faster than the present detection systems (which are using an enrichment step prior to testing). Implementing a real-time PCR system after capture would further reduce this detection time.

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