Progress 09/01/24 to 08/31/25
Outputs
Target Audience:The four target audiences were successfully reached and served during the grant period. They were as follows: (1) Undergraduate Students: Undergraduate students enrolled in the Foods (FOS 3026) lecture class were taught key food safety concepts. In addition, they received guidance on various career opportunities in the field of food science and food safety and were educated on how to find internships. Group meetings were held after each lecture with students enrolled in the food science program, during which mentoring was provided. This mentorship was particularly important, as many of these students are still exploring their options for graduate school and future careers. (2) Graduate Student: One graduate student was hired and trained in the areas of beef safety, pathogen detection, and the application of digital PCR assays to enhance beef safety. The student received extensive hands-on training with major foodborne pathogens, including Salmonella, Shiga toxin-producing E. coli, and Listeria. Additionally, the student was trained in both culture-based microbiological methods and molecular techniques such as real-time PCR and digital PCR. (3) Beef Industry Stakeholders: The Principal Investigator (PI) engaged with medium- and large-scale beef processors, conducting interviews to identify their challenges and research needs related to food safety. The PI gathered input on specific industry needs, particularly focusing on preferences for a Salmonella quantification method. These insights were integrated into the assay development phase of the project. (4) Large Food Testing Laboratories: The PI also reached out to experts from large food testing laboratories to collect input on their needs and preferences for Salmonella testing workflows in beef samples. These insights were instrumental in shaping the design and application of the assay. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Student Training and Professional Development: Recognizing that each student has a unique learning style, the PIprovided a variety of training and professional development opportunities tailored to undergraduate, master's, and Ph.D.-level students: Undergraduate Students: After each lecture, mentoring sessions were held to address student questions related to food science and careers in food safety. Students were given tours of the food safety laboratory and provided opportunities to interact with graduate students working on research projects. Master's Student: One master's student was hired to work on the research component of this grant. The student received extensive training in handling foodborne pathogens, with a focus on both culture-based and molecular detection methods. HAACP Training: A HACCP (Hazard Analysis and Critical Control Points) training program was organized on the Florida State University (FSU) campus. This training was made available to all food science students. Beef Processing Facility Visits: Two visits to beef processing facilities were arranged for the graduate student, providing firsthand exposure to commercial beef processing and real-world food safety practices. Industry Interaction: Zoom meetings were organized between graduate students and beef industry experts, offering students the opportunity to engage with professionals, gain insights into industry needs, and discuss potential career paths. How have the results been disseminated to communities of interest?Dissemination and Stakeholder Engagement: Initial results from the study were presented as an invited talk at the Southern Section AOAC Annual Meeting, held in Atlanta, GA. The presentation focused on the application of digital PCR technology for the detection of foodborne pathogens. In addition, meetings with beef industry stakeholders were organized through site visits and Zoom calls. These engagements served to update stakeholders on the progress of the research and to maintain active communication between the research team and industry partners. What do you plan to do during the next reporting period to accomplish the goals?Goals for the Next Reporting Period: Generate digital PCR assay validation data using laboratory-inoculated beef samples using a wide range of Salmonella serotypes, which are a concern for the beef industry. Compare the quantification results from the digital PCR assay with those obtained using the culture-based Most Probable Number (MPN) method. Explore options for patent protection of the Salmonella quantification assay developed in this study. Organize an FSPCA (Food Safety Preventive Controls Alliance) training program for food science students. Present a research poster and deliver an oral presentation at a food safety conference. Publish a peer-reviewed journal article based on the research findings.
Impacts
What was accomplished under these goals?
Salmonella is a globally recognized foodborne pathogen of concern. Although commercially available assays exist for the detection of Salmonella in food samples, most are limited to qualitative analysis (presence/absence test) and cannot quantify Salmonella levels. Recently, three assays were introduced for quantifying Salmonella in beef and poultry products; however, their release was accompanied by concerns regarding assay reproducibility in quantification results. Aim 1 of this project was to standardize a multiplex digital PCR assay for the simultaneous detection and quantification of Salmonella. For this aim, athree-target multiplex digital PCR assay was developed and optimized. The first primer-probe set targeted a conserved Salmonella genus-specific genomic region and was used for the detection and quantification ofSalmonella strains. The other two primer-probe sets were designed to specifically detect Salmonella Enteritidis and Salmonella Typhimurium, respectively. The concentrations of all three primer-probe sets, as well as the digital PCR amplification conditions, were optimized to ensure efficient and accurate multiplexing. The finalized assay was validated using pure cultures of target Salmonella strains and non-target bacterial strains. Assay validation steps were performed using a larger collection of DNA samplesand directly using whole-cell bacterial cultures. Results: Results from the multiplex digital PCR assay using 128 pure culture Salmonella strains demonstrated robust assay performance. The assay performed effectively with both DNA extracts and intact bacterial cells. It exhibited 100% specificity for the detection of the Salmonella genus, as well as for the Enteritidis and Typhimurium serotypes. The optimized assay did not show any cross-reactivity with any of the 24 non-target Entrobacteriaceae strains using DNA as well as whole cells. Aim 2 of this project was to validate the standardized multiplex digital PCR assay using laboratory-inoculated beef samples. To achieve this, the beef inoculation and enrichment processes were first optimized. In order to ensure optimal assay performance for quantifying Salmonella in beef samples, multiple DNA extraction protocols and commercial kits were evaluated for compatibility with ourtesting workflow. As a final step, a culture-based Most Probable Number (MPN) protocol was optimized to serve as a reference method for comparison with the digital PCR assay. Results: Buffered Peptone Water (BPW) was identified as the most suitable enrichment medium for enrichingSalmonella from beef samples. Enrichment samples were collected hourly, starting at the 5-hour time point and continuing through the 8-hour mark. Among the various DNA extraction kits evaluated, a custom-designed kit was determined to be the most effective for isolating DNA from beef enrichments. Initial testing using DNA extracted with this kit indicated that a 7-hour enrichment period was sufficient for the detection of Salmonella, while an 8-hour enrichment period provided better results for quantification purposes. The digital PCR assay closely quantified the inoculation amount of Salmonella added to the beef samples. The digital PCR assay further showed a DNA tolerance of up to 2000 nanograms of DNA per digital PCR reaction without any PCR reaction inhibition.
Publications
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