Location: Molecular Characterization of Foodborne Pathogens Research
Project Number: 8072-42000-093-000-D
Project Type: In-House Appropriated
Start Date: Apr 1, 2021
End Date: Mar 31, 2026
Objective 1: Development and validation of sample preparation methods for the detection of foodborne bacterial pathogens and toxins. Subobjective 1A: Generate, evaluate, and transfer a new class of magnetic materials for the effective partitioning and concentration of bacteria from large volume samples. Subobjective 1B: Adaptation of surface chemistry for the effective separation and concentration of pathogens from foods. Objective 2: Development and validation of rapid screening methods for foodborne bacterial pathogens and toxins, and identification of biomarkers. Subobjective 2A: Transfer methods to quantify foodborne pathogens. Subobjective 2B: Application of droplet digital PCR (ddPCR) to pathogen detection and quantitation. Subobjective 2C: Improve and expand the utility to aid in the transfer of the immunoelectrochemical biosensor technology for the detection of toxins and pathogens in food. Objective 3: Rapid identification, genotyping, and sequence analysis of foodborne bacterial pathogens. Subobjective 3A: Generate, evaluate, and transfer a novel AlphaLISA to confirm the presence of select foodborne pathogens. Subobjective 3B: Generate pathogen databases and improve the accuracy of the BEAM (formerly BActerial Rapid Detection using Optical scattering Technology or BARDOT) system. Subobjective 3C: Rapid identification and enumeration of both E. coli O157:H7 and Salmonella by MPN combined with multiplex qPCR. Subobjective 3D: Rapid identification of Campylobacter and Salmonella by target amplification and next generation sequencing using portable MinION sequencer. Subobjective 3E: Whole genome sequencing analysis of the phylogenesis, virulence factors and antimicrobial resistance of Campylobacter spp. from meat samples.
The primary goal of this plan is to develop rapid screening and identification methods for top, foodborne bacterial pathogens (Shiga toxin producing E. coli or STEC, Salmonella serotypes, L. monocytogenes, etc.). Testing for specific pathogens in select foods is sometimes an intermittent demand as gaps in methodology and needs may arise. However, the technology to be generated in this plan will proactively be suited for quick adaption to these needs typically only requiring, for example, substitution of a recognition element (e.g., antibody or DNA primer) or bioinformatics-based mining for unique stretches of DNA sequences. The detection of low levels of pathogens is complicated due to a gap in screening platform sensitivity, therefore we will increase sample volumes in order to elevate the amount of pathogens per test, especially when culture enrichment is not suitable (e.g., for rapid, field-based testing for very low concentrations of bacterial adulterants). To achieve this, novel sample preparation techniques will be key for rapid concentration of bacteria typically from aqueous homogenates. Subsequently, higher levels of detection sensitivity are expected as well as quantitation of extremely low levels (~1 cell/100 mL) of pathogens as needed for real-time testing. Assay times should be a few minutes to = 2 hours. Also, enhanced detection systems will be needed to bypass growth enrichment and achieve the desired detection levels. Furthermore, numerous biomarkers and the potential for false positive results using cross-reacting biorecognition elements (such as antibodies) will require multiplex detection techniques. However, for food contaminated with very low levels of target pathogens, detection may benefit from enrichment for accuracy thus avoiding false negative results. Therefore, conditions warranting brief enrichment prior to detection will be addressed. Methods will initially be developed with culture media or buffer as the sample matrix, and then extended to application with food (primarily ground meats). Assay performance of developed methods will be compared against “gold standard” methods initially with reliance on bacterial enumeration. Evntually, developed methods will be tested using FSIS samples in comparison to state-of-the-art methods. Yet the 5-year time frame for this plan may not allow for full scale, multi-laboratory validation of methods. Hence optimization of robust and reproducible technologies may better merit the time and financial investment associated with such validation. Eventually, testing will move to the field first off-line, then in-line (for some methods) in regulated environments. It is expected that multitudes of tests will be conducted given that most samples are negative for contamination by pathogens. Regulatory and perhaps legal guidance will be anticipated to be critical since validation testing will lead to remediation or recall if zero-tolerance organisms are detected or if certain instances of positive samples are discovered.