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We propose to adapt a highly sensitive and specific RT-PCR assay to detect heat-stressed but living L. monocytogenes. We will combine the latest resuscitation broth with this PCR assay to detect low levels of L. monocytogenes. This assay may then be used in a portable thermal cycler for rapid detection of this microbe in ready to eat foods. We will later propose to establish one state-of-the-art diagnostic laboratory which will be used to teach under graduate, professional and graduate students.
To determine the ability of the assay to detect the low levels of L. monocytogenes and other Listeria spp. in foods or other pathogens, ground turkey meat, will be irradiated to assure the absence of any Listeria spp. and inoculated with serial 10-fold dilutions of L. monocytogenes. The artificially contaminated product will be heated (15 min, 60 C). After heating an aliquot will be directly screened by the RT-PCR method without enrichment. Briefly, two primer sets will be used. Set I consists of primers Lis-1 and Lis-2. Set II consists of primers U1 and LI1. Primers Lis-1 and Lis-2 amplify a 174 bp region of the hlyA gene in L. monocytogenes. Primers U1 and LI1 target a 938 bp 16S rRNA sequence in members of the genus Listeria. A second aliquot will be placed in the one-step enrichment broth. At 4, 8, 16, 24, and 48 hrs, an aliquot will be removed from the enrichment and mRNA isolated and prepared for RT PCR analysis. In addition, a third aliquot of the heated turkey meat will be enriched in selective University of Vermont modified Listeria enrichment broth (UVM I) for 24 hours at 35 C and placed on to Columbia agar with 0.05% glucose supplemented with polymyxin B-acriflavine lithium chloride-ceftazidime-aesculin-mannitol (PALCAM) agar and incubated at 37 C for 24 hours for comparison of our method with conventional laboratory protocols. Assays will be validated using pure cultures and mixed cultures seeded into irradiated ground turkey meat. The specificity of the RT-PCR assay will be evaluated with the 11 serotype of L. monocytogenes and the six other recognized Listeria spp. including L. innocua. Specificity tests will also include few non-related bacteria associated with food-borne illness, including Clostridium spp., Campylobacter spp., Staphylococcus spp., Salmonella spp., Yersinia spp. and Escherichia coli. DNA extraction methods will be optimized in our laboratory (under the supervision of our USDA cooperator) and compared for efficiency and cost. These will include immunomagnetic bead separation, as well as boiling, diatom extraction, and use of commercially available nucleic acid extraction kits, such as Qiagen, InstaGene, and PrepMan.
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Listeria monocytogenes is a major food-borne bacterial pathogen of humans. Annually, it causes up to 2,500 human cases of severe disease including meningitis, encephalitis, sepsis, fetal death and premature birth. This agent has caused up to 500 human deaths annually. Only slaughtered animal (cattle, poultry and hogs) meats for human consumption have been known to be contaminated with this agent. It has been known that the consumption of pork and/or pork products has caused food-borne infection with Listeria in humans in North America and Europe. Healthy carrier hogs have been thought to contaminate the other unaffected animals. Development of a rapid and sensitive detection procedure and surveillance of L. monocytogenes and other Listeria spp. in pork, pork products and other livestock may aid in overall reduction and/or combating the human listeriosis. We propose to use a highly sensitive and specific Polymerase Chain Reaction (PCR) assay to detect heat-stressed but living L. monocytogenes and other Listeria spp. specifically in pork. We propose to establish a diagnostic laboratory, which will be used for both teaching and training in the use of different diagnostic methods to under-graduate, professional (D.V.M.), and graduate (M.S. and Ph.D.) students at Tuskegee University College of Veterinary Medicine, Nursing and Allied Health.