Project Number: 8072-42000-084-000-D
Project Type: In-House Appropriated
Start Date: Apr 1, 2016
End Date: Mar 31, 2021
1: Develop rapid and efficient techniques that separate and concentrate and/or quantify targeted pathogens from food matrices. 1A. Apply rapid and high volume centrifugal flow concentration to the separation of bacteria from food matrices. 1B. Partition and concentrate bacteria using immunomagnetic separation with a new class of antibody-coated paramagnetic particles. 1C. Compare and contrast bacteria separation and concentration with flow-through filtration systems. 1D. Develop and validate procedures for the rapid and quantitative detection of multiple foodborne pathogens. 2: Develop and validate field testing kits that rapidly screen for the presence and quantification of pathogens and/or indicator microorganisms in foods at the initial processing level. 2A. Generate portable, label-free sensors (e.g., next generation cantilever microbalance) for rapid in-line or near-line screening of foods. 2B. Generate portable antibody and/or phage-based multiplex assays including integrated comprehensive droplet digital detection (IC 3D). 2C. Develop an AlphaLISA detection protocol for target pathogens. 2D. Develop a flow-through immunoelectrochemical detection device for field portable detection of target pathogens. 3: Develop and validate rapid methods for the identification of pathogens and/or indicator microorganisms in foods for application in either the field or testing laboratories. 3A. Generate phage and/or antibody typing arrays. 3B. Generate pathogen databases and improve the accuracy of the Beam (formerly BActerial Rapid Detection using Optical scattering Technology or BARDOT) system. 3C. Direct typing (colony isolates not required) of enriched samples using a targeted-sequencing method. 3D. Generate genome sequence-based typing and identification schemes using next-generation sequencing technology (e.g., MiSeq, Ion Torrent PGM, and MinION), and characterize virulence and antibiotic resistance of microbial pathogens.
The primary objective of the plan is to develop rapid screening and identification methods for top foodborne bacterial pathogens, including STECs, top Salmonella serotypes, and Listeria monocytogenes as well as those of intermittent concern. Novel or enhanced sample preparation techniques (e.g., flow-through centrifugation, hollow fiber filtration, immunomagnetic separation), most likely in conjunction with pre-filtration, will be key for rapid concentration of food-associated bacteria to readily de detectable levels by modern rapid methods. Subsequently, improved levels of detection sensitivity are expected, perhaps even to an extremely low goal of approximately 1 cell/100 mL of target pathogen as required for real-time testing in the field, processing plant, distribution center, or retail establishment. Total assay times are foreseen to be from a few minutes to = 2 hours. Also, enhanced detection systems will be needed in order to bypass growth enrichment and achieve the desired, quantifiable detection levels. Furthermore, numerous biomarkers and the potential for false positive results using cross-reacting biorecognition elements will require multiplex detection techniques (e.g., multiplex qPCR and microarrays) that may be employed to distinguish true positive results from interference by background matrix or flora. Methods will initially be developed with culture media or buffer as the sample matrix, and then extended to application with food (primarily ground meat) in multiple sample formats: N=60 samples, meat core samples, tissue homogenates, carcass rinses, etc. The efficacy of any newly developed methods will require comparison to current “gold standard” methods in order to validate assay performance. Initially, this will be accomplished by reliance on enumeration of known bacterial isolates, quantified in pure culture with total cell counting if a significant dead population is expected. For evaluation, artificially inoculated and unknown samples will be tested with new methods as assessed against selective enrichment followed by selective and differential plate agar analysis. Regulatory-based methods, such as biochemical testing, multiplex PCR, and serotyping, and possibly whole genome sequencing, may be invoked for additional comparison. Our sister agency, FSIS, will provide guidance as to the parameters and specifics regarding acceptable validation of desired rapid bacterial detection methods. We propose that our developed methods be initially tested at FSIS regional labs using inspector obtained samples, split/divided at the lab, and tested in parallel. Eventually, testing will move to the field- first off-line and near-line, then in-line for some analysis platforms (e.g., microcantilever balance biosensor) situated in the processing environment and/or retail establishments. It is expected that multitudes of tests will be conducted given that most samples will be negative. Regulatory, and perhaps legal guidance will be anticipated to be critical since validation testing may lead to recalls if “zero tolerance” organisms are detected or if threshold amounts of positive samples (e.g., for Salmonella) are discovered.