Progress 09/15/00 to 09/14/04
Outputs
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.
Impacts
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.
Publications
- List of Publications and Presentations for 2004:
- Mathew, F. and Alocilja, E.C. 2005. Porous Silicon-Based Biosensor for Pathogen Detection. Biosensors and Bioelectronics Journal, 20(8):1656-1661.
- Mathew, F., Alagesan, D., and Alocilja, E.C. 2004. Chemiluminescence detection of Escherichia coli in fresh produce. Luminescence Journal, 19:193-198.
- Mathew, F. and Alocilja, E.C. 2004. Enzyme-based detection of Escherichia coli. Transactions of the ASAE, 47(1): 357-362.
- Mathew, F. and Alocilja, E.C. 2004. Porous silicon-based biosensor for pathogen detection, Poster presentation at the 8th World Congress on Biosensors, Granada, Spain, May 24-26, 2004.
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Progress 01/01/03 to 12/31/03
Outputs
RESEARCH COMPONENT Data analysis was conducted on the data generated from the previous experiments. A lot of time was spent on writing papers for peer-reviewed publication and presentation at professional meetings. A total of five papers were written and two presentations were made in 2003. Of the five papers, two were included in the proceedings (ASAE and IEEE); one paper has been accepted for publication and currently in press; another paper is in review; and another paper is being revised. The paper entitled Enzyme-Based Detection of Escherichia coli, which was submitted to the Transactions of the ASAE in late 2002 and revised in early 2003, was finally accepted for publication in December of 2003 and is currently in press. The paper entitled Rapid Detection of E. coli Using a Chemiluminescence Based Assay in Selected Produce was withdrawn from the Journal of Food Protection and is being revised for an alternative journal. Another paper entitled Chemiluminescence
Detection of Viable Escherichia coli in Fresh Produce Obtained from Different Sources was submitted to the Journal of Luminescence and is in review. Also, an alternative chemiluminescence assay made from porous silicon substrate was fabricated for use as an alternative chemiluminescence-based system. The new biosensor system will make use of silicon materials as the substrate. We are still in the initial stage of this new design. EDUCATION COMPONENT University Undergraduate Research and Arts Forum (UURAF): Two professorial assistants (members of the Honors College) were trained in the project. One professorial assistant presented her biosensor research during the 2003 UURAF. She was awarded second place for her poster presentation. Professorial Assistantship (PA) Training: Two professorial assistants (BE undergrads) were involved in the project. It provided early motivation to food safety and work in the laboratory. High School Honors Science Program Mentorship. Two high school
students from California and the Samoan Islands, participants in the 2003 High School Honors Science Program, were introduced to the project and mentored for 10 weeks in the the lab.
Impacts
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, providing the potential for a quick screening of microbial contamination in fresh produce.
Publications
- Mathew, F. and Alocilja, E.C. 2003. Chemiluminescence Detection of Coliform Bacteria In Fresh Produce Obtained From Different Sources. Paper #037065, ASAE Annual Meeting, Las Vegas, 28-30 July 2003.
- Mathew F. and Alocilja, E.C. 2003. Fabrication of Biosensor for Rapid Detection of Salmonella Using Porous Silicon Platform. Proceedings of the Second IEEE International Conference on Sensors, Toronto, Canada, 22-24 October 2003.
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Progress 01/01/02 to 12/31/02
Outputs
RESEARCH COMPONENT Samples of green leaf lettuce, parsley, spinach, strawberries, raspberries, alfalfa sprouts, and romaine lettuce were obtained from local grocery markets and used for the assay without any treatment, such as washing, that would alter the natural microflora. The samples were processed according to standard procedures for inoculating organisms, extracting, and testing for microbial loads. For all inoculated produce samples, the intensity of light emitted increased with the increase in bacterial numbers. In some cases, above 1000 CFU, the intensity of light emitted increased exponentially with bacterial numbers, as in parsley and sprouts, while exponential increase in light emitted was not observed for others, e.g., strawberry and green leaf lettuce. The assay sensitivity varied from produce to produce, e.g., for strawberry, sensitivity was 100 CFU while that of spinach was 1000 CFU. Parsley and raspberry had the best sensitivity of 10 CFU while
romaine lettuce had the lowest sensitivity of 10,000 CFU. EDUCATION COMPONENT Undergraduate Senior Design Instruction: Chemiluminescence technique developed from the project was taught to 4 Biosystems Engineering (BE) senior design students for the development of a rapid method for monitoring microbial contamination of sprout seeds. The students got training in microbial laboratory procedures and handling of biosafety hazardous waste, learned to conduct research and analyzed data, learned food safety issues and regulations of the food supply chain, and economics. The 4 BE senior design students participated in the 12th Annual International Environmental Design Contest (8-11 April 8-11 2002 in Las Cruces, NM) under Task 21: Alternative Sanitizers for Treatment of Seeds Used to Grow Sprouts. The students used chemiluminescence technique learned from the project to monitor the effectiveness of their sanitizer. The students designed and built a bench scale model of their sanitizer,
gathered and analyzed data, wrote a technical paper, and prepared a poster on their sanitizer. The students won the first place award. University Undergraduate Research and Arts Forum (UURAF): Two professorial assistants (members of the Honors College), trained in the project and presented their biosensor research during the UURAF on April 5, 2002. One student got first place award for oral presentation and the other got second place award for poster presentation. Professorial Assistantship (PA) Training: Four professorial assistants (BE undergrads) were involved in the project. It provided early motivation to food safety and work in the laboratory. High School Honors Science Program Mentorship. A student from Manhasset High School, New York, who participated in the 2002 High School Honors Science Program, was trained and mentored for 10 weeks to conduct chemiluminescence project in the lab. Graduate Special Topic Course Offering. The topic on biosensors was introduced to the BE
graduate students through BE 891 in the spring semester of 2002.
Impacts
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, providing the potential for a quick screening of microbial contamination in fresh produce.
Publications
- Mathew, F. and Alocilja, E.C. 2003. Enzyme-based detection of Escherichia coli. Submitted to the ASAE Transactions, in review.
- Mathew, F. and Alocilja, E.C. 2003. Rapid Detection of E. coli Using A Chemiluminescence Based Assay in Selected Produce. Submitted to the Journal of Food Protection, in review.
- Mathew, F. and Alocilja, E.C. 2002. Photon based sensing of pathogens in food. Proceedings of the IEEE Sensors Conference, Orlando, Florida, 12-14 June 2002.
- Mathew, F. and Alocilja, E.C. 2002. Rapid Detection Method for Coliforms Using a Chemiluminescence Based Assay. Proceedings of the ASAE Annual Meeting, Chicago, 28-31 July 2002.
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Progress 01/01/01 to 12/31/01
Outputs
RESEARCH COMPONENT Generic E. coli and E.coli O157:H7 cultures were maintained in trypticase soy broth containing 10 percent (v/v) glycerol and subjected to two consecutive overnight transfers in 9 ml of nutrient broth (NB). Polymixin B sulfate (45 microliters of 50mg/ml solution) was added to 9 ml of the above E. coli culture prior to incubation for 15 min. From this mixture, 25 microliters was then pipetted into a culture tube. After addition of 500 microliters Lumi-Gal 530, the tube was vortexed to mix well. Emitted light intensity was measured using a luminometer at 10 min intervals up to 60 min and two final readings at 75 and 90 min. Also, numbers of E. coli were determined by spread plating appropriately diluted samples on McConkey's agar. Bacterial counts were enumerated after 48 h of incubation. E. coli cultures were grown overnight in lactose broth (LB) as well as NB and assay procedure repeated as above in both cases. Also, light emitted by overnight LB E.
coli O157:H7 and generic E. coli cultures were compared. Following standardization of assay procedure, a Randomized Complete Block Design with 5 blocks of size 21 comprising a two-way treatment structure with 7 (dilution) and 3 (amounts of E. coli culture) levels was used to assess the sensitivity of the assay. The randomized trials were performed for 5 days to nullify the effect of minor day-to-day variation in bacterial counts. EDUCATION COMPONENT Undergraduate Senior Design Instruction: Chemiluminescence technique developed from the project was taught to 5 Biosystems Engineering (BE) senior design students for the development of a rapid method for detecting fecal contamination in fresh fruits and vegetables. The students got training in microbial laboratory procedures and handling of biosafety hazardous waste, learned to conduct research and analyzed data, learned food safety issues and regulations of the food supply chain, and economics. International Environmental Design Contest:
The 5 BE senior design students participated at the 11th Annual International Environmental Design contest under Task 3: Detection of fecal contamination in fresh fruits and vegetables. The contest was sponsored by Waste-management Education and Research Consortium and held on April 9-12, 2001 in Las Cruces, NM. The students used chemiluminescence technique learned from the project. Beta-galactosidase enzyme was used as indicator of E. coli contamination in fresh produce. The students designed and built a bench scale model, gathered and analyzed data, wrote a technical paper, and prepared a poster. In Las Cruces, the students presented their project to a group of 12 judges and demonstrated their bench scale operation. The students won first place in Task 3. University Undergraduate Research and Arts Forum (UURAF): Three of the five BE senior design students were members of the Honors College. The students presented their research results during the UURAF on April 6, 2001. The students
won second place. Professorial Assistantship (PA): One senior and one sophomore PA were involved in the project. It provided early motivation to food safety and allowed the PAs to work in the laboratory.
Impacts
Development of rapid detection method for microbial contamination in fresh produce will help reduce foodborne illness associated with the consumption of these products.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
This is a new project (started 9/15/2000). No progress to report at this time.
Impacts
(N/A)
Publications
- No publications reported this period
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