Fluorescence \u3ci\u3ein situ\u3c/i\u3e hybridization-based detection of \u3ci\u3eSalmonella\u3c/i\u3e spp. and \u3ci\u3eListeria monocytogenes\u3c/i\u3e in complex food matrices

摘要

Current methods for detection of Salmonella spp. and Listeria monocytogenes in food are culture-based methods and require performing numerous steps, between preenrichment, enrichment, selective plating, identification, and confirmation. Conducting these procedures can take several days; they require extensive manual labor and large amounts of media and reagents which can increase the cost of the testing. Molecular-based rapid high throughput methods can present a valid alternative to these methods, allowing for timely and sensitive detection of these bacterial pathogens before the contaminated products can reach the consumer, helping to prevent the occurrence of foodborne listeriosis and salmonellosis. Fluorescence in situ hybridization (FISH) is a sensitive and robust molecular method that uses sequence-specific rRNA-targeted fluorescently-labeled oligonucleotide probes to specifically label whole, permeabilized bacterial cells. When coupled with fluorescence microscopy or flow cytometry for analysis, FISH can be a powerful tool for detection of bacterial pathogens in food.My hypothesis was that we could develop rapid and sensitive FISH-based methods for detection of these two pathogens in complex food matrices. My research concentrated on four objectives: 1. Optimize use of existing FISH probes and hybridization conditions for detection of Salmonella spp. and Listeria monocytogenes; 2. Develop pre-anayltical food sample preparation methods compatible with downstream approaches for whole-cell detection; 3. Utilize the results of objectives 1 and 2 to develop FISH-based assays for detection of Salmonella spp. and L. monocytogenes in foods; and 4. Establish the ultimate detection sensitivity of the developed methods.Specifically, optimal combinations of existing Salmonella-specific probes were developed and applied for rapid (15 min) hybridizations of target cells. Use of these probe cocktails was integrated with pre-analytical sample preparation steps, including tangential flow filtration, adhesive tape sampling and immunomagnetic separation to enable sensitive detection of Salmonella spp. in complex food systems via flow cytometry or fluorescence microscopy. The food systems studied included alfalfa sprouts, fresh produce (tomatoes, jalapeyo peppers, spinach, cilantro), and peanut butter. Pre-analytical sample preparation using pulsification also improved the signal-to-noise ratio for cytometric detection of Listeria monocytogenes in pork frankfurters via flow cytometry following FISH. In addition, use of the PulsifierTM enabled detachment of surface-bound L. monocytogenes cells into minimal volumes of diluent, obviating the need for the subsequent cell concentration steps typically required prior to detection.Results from this work suggest that, when paired with effective methods for upstream food sample preparation and with downstream analytical methods such as flow cytometry and fluorescence microscopy, FISH-based methods have great potential for rapid molecular detection of Salmonella spp. and L. monocytogenes in foods.