Ulta-Sensitive and Rapid Detection of Listeria Monocytogenes in Ready-to-Eat Food Products with Nonfluidic Array System

NON-TECHNICAL SUMMARY: A rapid, reliable system for detection of LM from RTE products is an important tool to undertake further studies on the epidemiology of this pathogen in humans and to control its presence in foods. With a strong identification methodology, scientists will be able to test different intervention technologies aimed at reducing the number of LM during food production. LM is a ubiquitous organism and is capable of growing at refrigeration temperatures. Therefore, the zero-tolerance ruling issued by the U. S. Department of Agriculture for LM in RTE foods presents a serious challenge to the food industry. Studies have demonstrated that RTE products acquire LM contamination from the processing environments. Because no further heat inactivation step is applied to RTE products before consumption, these products are of particular concern to the industry. By meeting the USDA goal of delivering safer foods by enhancing the detection of foodborne pathogens, the results obtained from this project will be used to submit grant proposals that will be able to secure extramural funds from federal agencies to further expand its application. The limitations of the current methodology for identification of LM is low sensitivity and the time needed to obtain the results. Most of the methods require an enrichment of at least 12 to 18 h to allow LM cells to multiply to detectable levels. Therefore, the development of a ultrasensitive and reliable methodology for rapid identification of LM in RTE products is of great benefit to the food industry and health professional.

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APPROACH: Primer Set and Probe for the Molecular Marker: The proposed research could be able to detect single LM cells though fast genetic amplification of specific DNA targets with the polymerase chain reaction (PCR) on a microfluidic chip. With the intrinsic characteristics of microfluidics, fast reaction and high sensitivity could be achieved. This approach requires specific primer sets to amplify the target gene of the hemolysin gene (hly) to distinguish LM from other similar Listeria spp., mainly L. seeligeri. Fluorescently labeled probes will be used to address the amplification of the target gene. The hly gene encodes a pore-forming hemolysin, listeriolysin O (LLO), which is essential for pathogenicity. Several sets of primers will be tested, and the most efficient set will be used for the fast amplification of hly gene with single template sensitivity on the nanoliter fluidic arrays. Specific fluorescent-labeled probes will be designed and used to report the amplification of the hly. Once the best primer set and the probe are chosen, LM cells will be mixed with PCR cocktails and off-chip detection of LM will be carried out using a real-time PCR (Light Cycler, Roche Diagnostics). To verify the target amplification specificity, PCR products will be sequenced with an ABI 3100 Genetic Analyzer (Applied Biosystems, the Auburn University Genomics & Sequencing Lab). Sequences will be compared for similarity using the nucleotide-nucleotide search feature of BLAST (Basic Local Alignment Search Tool; http://ncbi.nih.gov/BLAST/). If primers targeting the hly gene react with L. seeligeri isolates, an alternative set of primers that target a fibronectin-binding protein-encoding gene (fbp) that is specific for LM and L. welshimeri will be utilized. Detection of Listeria Cells in Nanofluidic Systems: The specificity of the primers in detecting multiple strains of LM will be confirmed using a conventional real-time PCR. These primers will be incorporated into the nanoliter fluidic arrays coupled to a modified thermal stage. First, the sensitivity of detection will assessed with pure cultures of LM. Then, the system will be tested with food matrices (i.e., ready-to-eat products. such as deli meat and hot dogs). Inoculated products will be processed (i.e., filtration, centrifugations etc) to separate the LM for subsequent detection trials. Solutions containing the LM cells will be loaded onto the microfluidic chip for theoretically single cell LM detection. Fluorescent probes will be used to verify the presence of LM using a high-resolution bioarray detection system that was modified for microfluidic applications.

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PROGRESS: 2006/10 TO 2009/09<BR>
OUTPUTS: Through this project, a solid research collaboration has been established between laboratories from the Department of Poultry Science and Materials Research and Education Center of Auburn University. During the course of this project several researchers, graduate and post-doctorate students were exposed to the application of nanotechnology to food microbiology. <BR> PARTICIPANTS: Individuals on the project consisted of S.F. Bilgili (PI), Kenneth S. Macklin, and Omar A. Oyarzabal from the Department of Poultry Science, College of Agriculture and Jong Wook Hong from the Alabama Microelectronics Science and Technology Center, and Nanofluidics Laboratory, Samuel Ginn College of Engineering,Auburn University. Throughout the project, training opportunities were utilized for graduate and post-doctoral students. <BR> PROJECT MODIFICATIONS: One nanoliter fluidic arrays (NFA) chip containing 10,000 nanoliter fluidic chambers with 10 different sample pads have been successfully fabricated. Due to evaporation problems, the filling reagent for control channels were changed to polyethylene glycol (PEG) solution to provide for better thermal stability during operation. Due to the increased surface-to-volume ratio, a major difficulty was to keep the thermal stability of liquids in the order of 5 nanoliter volume at 95C. This temperature is necessary to start and perform polymerase chain reaction (PCR) assays for the detection of the target DNA. Our efforts continue to achieve advanced chip design and fabrication for thermal stability for PCR assays.
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IMPACT: 2006/10 TO 2009/09<BR>
Different nanoliter fluidic arrays were refined (i.e., designed and fabricated) to be able to detect a single cell of important food-borne pathogen L. monocytogenes with a real-time PCR. When validated, this technology should allow unprecedented sensitivity in bacterial detection on fully-cooked meat products and provide for safer food supply. Knowledge gained from this project allowed the securing of additional funding.
<BR> <BR> PROGRESS: 2007/01/01 TO 2007/12/31<BR>
OUTPUTS: Jong Wook Hong, Abstract of the annual meetings of the International Association of Food Protection (IAFP 2007), "Nanoliter Fluidic Arrays for Single Bacterial Cell Detection", July 8-11, 2007, Lake Buena Vista, Florida <BR> PARTICIPANTS: Leslie Speegle (MS student) and Scott Miller (MS student) <BR> TARGET AUDIENCES: The food processing industry needs to have access to new methods that simplify the technique for rapid analysis of food samples to determine the presence of Listeria monocytogenes in food. <BR> PROJECT MODIFICATIONS: Progress Statement: Six primer sets (S1-S6) were designed from the sequence of the hemolysin gene of L. monocytogenes. One set of primer (S1) had a better specificity and sensitivity than the rest. A set of primers was also designed for the amplification of the gene coding for the internalin surface protein InlB of L. monocytogenes. The InlB primer was very specific and only produced amplified product in L. monocytogenes strains. However, the sensitivity was not as good as the SI primers. We are currently using real-time PCR to miniaturize the reaction volumes to 5 micro litter or less and to validate the efficacy of these primers for the microfluidic detection device. A nanoliter fluidic array chip containing 10,000 nanoliter fluidic chambers with 10 different sample pads was successfully fabricated. Although the micromechanical valves in the nanoliter fluidic array chip were functioning properly, evaporation problems with water-filled fluidic channels during thermal reactions made us change to polyethylene glycol solution to provide better thermal stability.
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IMPACT: 2007/01/01 TO 2007/12/31<BR>
New insights have been gained on the thermal stability of small amount of liquid (1 nanoliter). Several different nanoliter fluidic arrays have been designed and fabricated in the Alabama Microelectronics Science and Technology Center and the Nanofluidics Laboratory in Materials Engineering at Auburn University. Yet, more options will be tested in the coming year.