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Chemiluminescence Detection of Microbial Contaminants on Fresh Produce


<ol> <li>To validate and apply the membrane immunoassay chemiluminescence (MIC) detection system developed at Michigan State University to rapidly detect pathogens, such as enterohemorrhagic E. coli (EHEC) O157:H7 and Salmonella typhimurium on contaminated fresh produce;
<li>To extend the techniques learned from research to senior design instruction and undergraduate competition and research forum;
<li>To use the research project as training for Honors College BE students, professorial assistants, UURCAF participants, and high school students in HSHSP; and 4. To design and develop a bench scale prototype for feasibility testing with partnering industry.</ol>

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The methodology consists of three components: research, education, and technology transfer (extension).
The research component involves several activities, namely: <ol> <li>Set-up the MIC biosensor;
<li>Pure culture preparation;
<li>Chemiluminescence measurement in pure culture;
<li>Generation of dose-response curve for each target analyte;
<li>Sample preparation;
<li>Microbial analysis;
<li>Chemiluminescence measurement of spiked samples; and
<li>Chemiluminescence measurement of fresh samples.</ol>
The education component involves food safety and rapid detection training for high school, BE and Honors College students, professorial assistants, and graduate students; participation in the environmental design contest and undergraduate research forum; and development of a special topic course on food safety and rapid detection systems. The extension and technology transfer component includes the development of a bench scale for testing at Neogen Corporation, along with the development of operating manual consistent with food safety, industry, and labor regulations. Bench scale testing will pave the way for the feasibility of commercialization of the instrument. Partnership with Neogen Corporation will facilitate transfer of technology to the fresh produce industry, thus protecting consumer health. Duration of the project is three years.
Objective 1: During the early stages of project implementation, strains of Salmonella Typhimurium DT104 and Escherichia coli O157:H7 were used to test the performance of the membrane immunoassay chemiluminescence (MIC) detection system as outlined in the proposal. We had initial success but encountered difficulty in making the results reproducible and consistent. To be effective in the field, these factors needed to be addressed. Thus the original procedure was modified using (a) beta-galactosidase enzyme to replace the hydrogen peroxidase enzyme and (b) dioxetane to replace luminol, in the chemiluminescence reaction as outlined in the original procedure. Beta-galactosidase is an enzyme produced by E. coli as part of its regular metabolic process. To provide specificity to E. coli O157:H7 in the detection process, antibodies specific to the strain were used to initially capture the organisms in the sample. The application of the assay was successfully demonstrated using spiked samples of food and water. The modified assay was used to test the presence of E. coli O157:H7 in artificially contaminated water, milk, apple cider, and 60 samples of fruits and vegetables collected from various sources. The detection limit of the assay was found to be from 100 to 1000 CFU. The assay was completed within 60 min. A nanoporous silicon membrane was also developed as an improved surface to replace the cellulose membrane as outlined in the original proposal. The nanoporous silicon membrane was successfully developed and tested for Salmonella detection following the original procedure using antibody-hydrogen peroxidase conjugate with luminol.
Objective 2: The chemiluminescence biosensing technique was taught to a total of 15 Biosystems Engineering senior design students over a period of three years for the development of a rapid method for detecting fecal contamination in fresh fruits, vegetables, and water. Three teams (Year 2000, 2001, and 2002) were funded through this project and were successful in competing at the International Environmental Design Contest held annually in Las Cruces, New Mexico. All three teams won first place in their respective tasks.
Objective 3: A total of 10 professorial assistants, Honors College students, and engineering student interns were trained through this project. They presented their research work at the annual University Undergraduate Research and Arts Forum. A total of 10 HSHSP students were mentored and trained through this project. Three of the high school students received national awards for their research projects.
General Objective 4: Although we encountered administrative problems in testing the protocol in the laboratory of our partnering industry, we were still able to accomplish this objective. We used the senior design students in 2003 as surrogate for industry personnel. We provided the design and testing protocol to the students and the students implemented the method in their own senior design project for developing a rapid detection system of E. coli in a drinking water system. The students were successful in following the protocol and in finishing their project.
Dietary recommendations of leading health authorities encourage consumption of five or more servings a day of vegetables and fruits, all year round, in order to reduce the risk of heart diseases as well as cancer. However, at the same time, fresh produce has been determined to be the vehicle of transmission for several foodborne disease outbreaks, potentially because fresh produce undergoes minimal processing and they are more exposed to areas of contamination. Enzyme-based chemiluminescence assay provides a simple and rapid method for the detection of viable coliforms and bacterial pathogens, providing the potential for a quick screening of microbial contamination in fresh produce.

Alocilja, Evangelyn
Michigan State University
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