The overall goal of the project is to integrate food pathogen detection laboratory research and education with the ultimate aim to improve food safety. This integrated multi-disciplinary proposal is specifically targeted to translate foodborne pathogen detection methods developed in our laboratory (research) into augmenting the knowledge base about food safety by training minority students in applied food safety (education).
<P>The proposed project will have the following specific goals: i) Develop nucleic acid-biosensor -based detection assays which will have simultaneous or multiplexed detection capacity, ii) Train, motivate, mentor and develop skilled manpower in food safety, foodborne pathogen detection technologies among the underrepresented minorities, iii) Enhance the capacity of the faculty in the College of Veterinary Medicine, Nursing and allied Health (CVMNAH) to stay abreast with advances in foodborne pathogen detection technologies and the impact of food safety issues. The research arm of the project we will integrate our specific real time- PCR based foodborne pathogen detection tools and antibodies with impedometric biosensor technologies and hand-held surface plasmon resonance (SPR), respectively. <P>The education component of the research will enable students to be trained in the genomic bioinformatics and the development of the biosensor technologies stated above, and for the faculty to update their knowledge through attendance and presentation at relevant meetings. <P>The measurable outcomes will be a) Graduate two students (MVSc) with theses focused on integrated detection of food borne pathogens, b) Improve the contents of two elective courses offered to graduate students at CVMNAH, by including current advances and tools for the molecular and biosensor-based detection techniques of foodborne pathogens, c) Active participation of project members at national meetings relevant to food safety and food microbial biodetection. Successful development, implementation, testing, and validation of the proposed methodologies will minimize an existing gap in our capacity to rapidly detect multiple pathogenic threats simultaneously in real time. We anticipate that this collaborative research will provide cross-disciplinary experience to the PI's of the project with respect to detection technologies (foodborne pathogen detection, genomic bioinformatics, PCR, nanotechnology, biosensor development). Additionally, it will encourage minority veterinarians to undertake post-DVM education in food safety industry and pursue research-oriented careers. The state-of-the-art research will boost the confidence of CVMNAH graduates and is expected to improve their marketability for jobs in the federal government as well as in industry. The resources developed through funding for this project will enhance the quality of research by graduate students and faculty. Project funds will train minority graduate students who subsequently will contribute to work force diversity in the state, federal agencies and in the industry.
Foodborne pathogens continue to cause epidemic illnesses and immense economical damage to the US industry and the public. The number of cases of gastroenteritis associated with food is conservatively estimated to be between 68 million and 275 million per year. Lacks of rapid, sensitive and susceptible foodborne pathogen detection assays may exacerbate the issue and also result in under-reporting of foodborne illness outbreaks. The Food Safety and Inspection Service (FSIS) within the USDA inspects and regulates meat, poultry and processed egg products produced in federally inspected plants. Despite the existing efforts to ensure food safety, massive outbreaks continue to happen with enormous health, economical and social consequences. There exists local and national need, among many others to: a. Develop workforce at national level in the areas of genomic data mining and incorporation of microbial genomic data into the pathogen detection tools and platforms. b. Rapidly and reliably detect foodborne pathogens in food before or as soon as outbreaks of infection are reported, c. Develop rapid, sensitive and specific detection strategies to diagnose foodborne illnesses, d. Train students (mainly minority) and laboratory personnel at Tuskegee University on the recent advances in foodborne pathogen detection platforms and molecular technologies, especially those that have field applications. This proposal addresses these issues by attempting to develop hand-held and laboratory-based detection tools, as well as by training minority students in methods in food safety molecular technologies. Traditional methods for foodborne pathogens detection are time consuming and laborious, so there is a need for innovative methods that enable rapid identification of foodborne pathogens. Recent advances in molecular techniques have revolutionized the detection of pathogens in foods. The advent of biotechnology has greatly altered food-testing methods. In this study we will develop PCR-based technique and surface plasmon resonance (SPR) biosensor application for the rapid identification of the foodborne pathogens (Campylobacter, Salmonella, E. coli and Listeria). The overall outcome of this project will be to translate pathogen detection methods developed in our laboratory and validate them at a point of concern.
Foodborne pathogens included in the research for single and multiplex PCR will be Salmonella, E. coli, Campylobacter and Listeria. As this will be the first study of its kind, we will establish the Surface Plasmon Resonance (SPR) technology for the detection of only two foodborne pathogens: E. coli and Salmonella species. Commercially available anti-Salmonella (Abcam #31555) and anti-E. coli (Abcam #25823) antibodies will be used. As these antibodies have already been characterized and well validated for ELISA assays, we can directly immobilize them on the protein-G chip surface. Once the system is established, the SPR device could also be further modified to detect other pathogens or agents. Reference strains will incorporated in each validation.
<P>Approach: Nucleic acid-based detection assay:<br/> In our previous work, we validated the strain specific primers and probes for most of the selected foodborne pathogens. We will identify appropriate amplification parameters for the Salmonella subspecies that we had not addressed in our previous work. These will be combined with the probes we already identified, to establish the PCR microarray. The probes will be initially validated in-silico, and then in vitro using isolated bacterial genomic DNA. In our previous work, a number of published sequences showed cross-reactions with related or distantly related bacteria when tested against a larger pool of genomic data (in silico) and bacterial genomic DNA (in vitro). Therefore, every probe we will synthesize will be vigorously tested against about 30 genomes of species and strains of pathogens for cross reactivity b. Label free SPR biosensors: On-Site Monitoring of Foodborne Pathogens Using Hand-Held Surface Plasmon Resonance (SPR): SPR-based technologies have the advantage of simplicity and adaptation for field use. In this experiment, main focus will be on the development of SPR-based immunoassays for bacteria detection, concentrating on instrumentation, surface functionalization, liquid handling, and surface regeneration. Moreover, SPR-based pathogen detection devices are now available in hand-held formats, which we can modify by functionalizing the surfaces with our own probes and use for the detection of the pathogens listed above. In this proposal, for orientation-controlled immobilization of antibody onto a SPR surface, protein G modified with cysteine residues will be used as antibody-capturing molecule, since protein G selectively binds to the heavy chain constant Fc region of antibodies. Publication and presentation and practical application of the product to the user will be used as output evaluation. Arrangements will be done once in 6 months to meet all the co-investigators and collaborators to evaluate the progress and future directions of the project. The experimental design and data analysis will be supported by the Center for Computational Epidemiology, Bioinformatics & Risk Analysis (CCEBRA) and Biomedical Information Management Systems (BIMS) located in the College of Veterinary Medicine, Nursing and Allied Health.