PROJECT 1: Bioreporter-based technology for detection of organic toxicants directly from milk and milk products <BR> Objective 1.1 Develop promoter-reporter gene constructs for detection of BETTX (benzene, ethylbenzene, toluene, trichloroethylene, and xylene) and PCBs. <BR> Objective 1.2 Standardize bioreporter assays for detection of BETTX (benzene, ethylbenzene, toluene, trichloroethylene, and xylene) and PCBs in milk and milk products. <BR> Objective 1.3 Survey milk and milk products for BETTX (benzene, ethylbenzene, toluene, trichloroethylene, and xylene) and PCBs along the food chain continuum. <BR> <BR> PROJECT 2: Molecular beacon-based technology for detection of bacterial pathogens directly from milk and milk products <BR> Objective 2.1 Develop and standardize molecular-beacon based real-time PCR assays for , Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, Salmonella spp., Staphylococcus aureus, Bacillus anthracis and Mycobacterium avium subsp. paratuberculosis. <BR> Objective 2.2 Survey milk and milk products for bacterial pathogens along the food chain continuum using molecular beacon-based real-time PCR assays.
NON-TECHNICAL SUMMARY: Develop state of the art diagnostics for detection of organic toxicants and foodborne pathogens in milk and milk products This project will focus on developing reliable and highly sensitive assays for detection of organic toxicants and foodborne pathogens in milk and milk products that can be used for monitoring and safeguarding milk supply and milk products.
APPROACH: <BR> PROJECT 1: The toluene degradation operon (tod) that degrades benzene, ethylbenzene, toluene, tricloroethylene, and xylene (BETTX) will be amplified from the genomic DNA of the Pseudomonas putida (ATCC53902). The primers for the PCR amplification for the tod promoter and regulator elements will be designed using the tod sequence reported by Mosqueda and Ramos (2000). The biphenyl dioxygenase gene (bphA1) has been shown to degrade PCBs. The gene will be amplified from genomic DNA of the Burkholderia cepacia (ATCC 55487). The primers for PCR amplification for the bphA1gene will be designed using the bphA1gene sequence (AJ251217) in the NCBI nucleotide database. The PCR amplified product will be cloned in to pGlow-TOPO vector (Invitrogen, Carlsbad, CA) containing green fluorescent protein (GFP) as reporter. This individual plasmid constructs will then be introduced in to E. coli TOP10 (Invitrogen) by electroporation (BioRAD micropluser, Hercules, CA) for use as a bioreporter. The transformed E. coli strains will be tested rigorously by exposing the strains to specific toxicants (concentration ranging from 10 microMol to 1 mMol) suspended in buffered peptone water. Expression of GFP proteins by bioreporter E. coli strains will be measured by reading the intensity of fluorescence in a spectrofluorimeter at intervals of 90-, 120-, 180-, 210-, and 240 minutes of incubation. <BR> <BR> PROJECT 2: Molecular beacons, PCR primers and PCR conditions that will be used to develop the assays will be from techniques reported in literature for C. jejuni (Sails et al., 2003), E. coli (Fortin et al, 2001), L. monocytogenes (Nogva et al 2000), Salmonella (Chen et al., 2000), B. anthracis (Verma-Basil et al., 2004), and M. avium subsp. paratuberculosis (Rodrigeuz-Lazaro et al., 2004). The assays will be standardized using control molecular beacons, bovine cellular DNA and common milk bacterial DNA as internal standards. The molecular beacon-based PCR assays will be developed and standardized individually for all seven organisms. <BR> <BR> ASSAY APPLICATIONS: Milk and milk products, milk filters and colostrum will be collected and analyzed for organic toxicants and C. jejuni, E. coli, L. monocytogenes, Salmonella, S. aureus, B. anthracis and M. avium subsp. paratuberculosis. Raw milk from 10 dairy producers that supply milk to the processing plant will be traced along the food continuum (farm---milk hauler---milk silo---pasteurized milk in retail markets). Samples will be collected over a period of 6 months from the farm, milk trucks, milk from the processing plant, and pasteurized milk sold by the processing plant in the retail markets. In addition, cheese (domestic and imported), evaporated and condensed milk, non fat dry milk from retail stores will also be examined for bacterial pathogens. A total of 10 dairy herds will be selected (milk collected by the same milk hauler and on the same milk route) and samples (milk filters, bulk tank milk, colostrum) will be collected once every 2-3 weeks by a trained technician.
PROGRESS: 2005/08 TO 2007/07<BR>
OUTPUTS: Key Outputs <BR> 1) Vectors that encode for organic toxicants were developed and successfully tested. These vectors are available for other researchers to test and validate the system <BR> 2) Real time PCR assays were developed for important foodborne pathogens. These RT-PCR assays have been validated through a multi-laboratory diagnostic system and currently in use in three laboratories. <BR> PARTICIPANTS: Bhushan Jayarao, Principal Investigator; Coordinate project and report results. Dr.David Wolfgang, Co-Investigator; Oversee sample collection and processing. Dr. Key, Co-Investigator; Advice on developing molecular beacon PCR assay. Dr. Perdew, Co-Investigatgor; Advice on PCB analysis. Dr. Brenda Love, Co-Investigator; Advice on bacteriological analysis of foodborne pathogens. Dr. DebRoy, Co-Investigator; Advice on molecular beacon PCR assays. Dr. Heinrichs, Co-Investigator; Identify and solicit participation of dairy herds. Dr. Narsimha Hegde, Post doctoral research associate; Develop, test and standardize bioreporter assays. Ms. Beth Houser, Research Technician; oversee day to day operations of project. Ms. Sarah Donaldson, Research Technician; conduct molecular beacon assays. <BR> TARGET AUDIENCES: Animal Diagnostic Laboratories, Food Testing Laboratories, Federal, State and Local government agencies with focus on food safety Food Safety Experts <BR> <BR>
IMPACT: 2005/08 TO 2007/07<BR>
1) The bioreporter system developed can be used for screening large number of samples, in the event of accidental or intentional contamination of milk and milk products.<BR> 2) The multiplex RT PCR system has been developed for use in veterinary and food diagnostic laboratories.