Description:
OBJECTIVE: To develop a field-ready kit for the rapid (max 8 hours) identification, quantification, and viability of microbial pathogens (bacterial, viral, and eukaryotic) from food matrices, water, and environmental samples. Direct or indirect detection of biological toxins is also desired. A developed kit will emphasize ease of use by technicians who are relatively lab-inexperienced, and an agnostic/semi-agnostic approach to sample setup and testing. DESCRIPTION: The United States Air Force public health and laboratory career fields have few field-capable diagnostic tools to identify microbial pathogens from food samples suspected of causing illness. Current capability relies on the non-differential and non-selective PetrifilmsTM (3MTM) that are incubated overnight to produce a total aerobic plate count; separate steps that incorporate the use of crystal violet dye or a heating step also allow the separate enumeration of Gram-negative and spore-forming bacteria, respectively. However, the resulting data is inadequate for deciding the appropriate course of action in determining the source and/or preventing the spread or reoccurrence of a Food-Borne Illness (FBI) event, as this kit does not provide enough data required for the unambiguous ID of a microbial pathogen. The initial organisms of interest are the top ten pathogens responsible for the vast majority (>95%) of food-borne illness events in the United States as identified by the Center for Disease Control (Scallan et al. 2011). They are: 1. Norovirus, 2. Salmonella spp., nontyphoidal, 3. Clostridium perfringens, 4. Camplyobacter spp., 5. Streptococcus spp. Group A, 6. Shigella spp., 7. Escherichia coli O157 and non-O157 spp., 8. Yersinia enterolitica, 9. Toxoplasma gondii, 10. Giardia intestinalis. The primary user of the developed kit will be USAF public health officers and technicians with little or no laboratory training under potentially extreme environments without access to ideal laboratory conditions and facilities. The kit must demonstrate cost-effective and user-friendly assays under field conditions with same-day (eight hours maximum) results at the genus or species level of identification from food samples. The kit must have agnostic (i.e., one assay can identify all ten initial organisms) or semi-agnostic (assays are broken into classes such as Gram-positive/Gram-negative/virus/parasite) sample preparation in order to reduce the number of tests per sample, save time, and keep costs low. PHASE I: Key deliverables: 1. A technology that can detect and identify microbial pathogens with same day results (~eight hours maximum) from food, water and environmental samples. 2. Quantitative or semi-quantitative results. 3. Agnostic or semi-agnostic sample preparation and assay set-up. 4. Ease of use; must not require extensive laboratory experience or training. 5. Process will include all steps from initial treatment of sample to readout of result. PHASE II: Key deliverables: 1. Technology from Phase I ruggedized for field use. 2. Cost-effective equipment; inexpensive and shelf-stable consumables/reagents. 3. Small footprint (
