Performing Department
(N/A)
Non Technical Summary
The presence of Salmonella is one of the biggest threat to the global food industry. Each year, Salmonella contamination results in millions of human infections. The quantitative PCR is the most common technology used for testing Salmonella in food samples. Advancements in digital PCR technology are enabling the development of assays that can detect Salmonella and, at the same time, quantify Salmonella load in food samples. The digital PCR assays are more robust, can tolerate large quantities of crude DNA, can generate reproducible results in the presence of PCR inhibitors, and can quantify Salmonella load in test samples. Samples contaminated with high Salmonella levels, Salmonella Enteritidis, and Typhimurium have a higher risk of causing severe human infections. Therefore, this project aims to develop a method to detect Salmonella contamination in beef samples, quantify the Salmonella load, and identify if the samples are contaminated with Enteritidis and Typhimurium serovars.The project's overall goal is to perform applied research in the area of Food Science and develop a diagnostics assay needed by the beef industry, provide training to graduate and undergraduate students, and expand Florida State University's research capacity for performing food safety research. The intended beneficiaries will be students, faculty, participants, the beef industry, academia, and the public. The expected outcomes are a novel digital PCR assay for Salmonella detection and estimation, peer-reviewed publication, presentations to academia and industry stakeholders, student training and undergraduate experiential learning, increased student knowledge of new diagnostic technology, workshops on food safety, and development of communication, leadership, critical thinking, and problem-solving skills among participating students.The availability of a standardized through this grant will serve the diagnostic needs of a wide user base, which includes small and large food processors, federal laboratories, and third-party testing laboratories, and generate trained students, enabling us to achieve a broader impact and thus improve public health.
Animal Health Component
25%
Research Effort Categories
Basic
(N/A)
Applied
25%
Developmental
75%
Goals / Objectives
Salmonella strains that are pathogenic to humans contribute to1.35 million cases annually. One overlooked issue associated with the high number of Salmonella infections is that the commonly used testing approach relies on "presence/ absence" testing of food enrichments and ignores the pathogen concentration in the contaminated samples. Knowing the Salmonellaconcentration in food samples and the Salmonella serovarsis crucial for making informed regulatory decisions. The goalof our researchis to accelerate the development of robust foodborne pathogen detection and estimation workflows that can be adopted by regulatory agencies and other food testing laboratories. The objectives of this project are as follows: The objective one is to optimize amultiplex digital PCR assay for absolute quantification of Salmonella and simultaneous identification of contaminating Enteritidis and Typhimurium strains. The objective two is to validate the standardized multiplex digital PCR assay using laboratory-inoculated beef samples.
Project Methods
Quantitative PCR-based assays are the most commonly used for testing pathogens in food samples. Over the last decade, digital PCR technology has significantly evolved, and now digital PCRcompletely outcompete quantitative PCR technology. Our approach for the development of a multiplex assay for the detection and quantification of Salmonella will involve the selection of the best-suited industry-friendly method for crude DNA extraction, DNA concentration step, and optimization of Salmonella genus-specific probe, Enteritidis and Typhimurium-specific hydrolysis probe, optimization of multiplex digital PCR assay, followed by assay validation with pure culture strains, and laboratory-inoculated beef samples. The assay results will analyzed following USDA guidelines for assay developers and AOAC Appendix J. The quantification value achieved by the digital PCR assay and the Salmonella inoculated to the beef samples will be statistically comparedwith a one-sample t-test.