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Integrating Detection of Multiple Pathogens in Food

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  • 1. Lawrence Goodridge Department of Animal Sciences Colorado State University [email_address] Integrating Detection of Multiple Pathogens in Food
  • 2. Introduction
    • Cultural methods
      • Slow (require 24 -48 hours or longer for results)
      • Can’t detect viruses or toxins
    • Immunological methods
      • Specific, moderately sensitive
      • Slow (require 24-48 hours for results)
      • Labor intensive
    • Molecular methods
      • Very specific, sensitive
      • Expensive
      • Require operator training
  • 3. Objective
    • To develop a multiplex assay for rapid detection of the foodborne pathogens Listeria monocytogenes, Salmonella spp., shiga toxin producing Escherichia coli, and fecal coliforms as indicators of fecal contamination
  • 4. Biochemical Assays Previously Developed Salmonella Listeria monocytogenes Escherichia coli
  • 5. Disadvantages of the Biochemical Assays
    • Still too labor intensive
    • Sensitivity and substrate issues
    • Can only detect one microorganism at a time
  • 6. Paper-Based Testing Devices
    • Fluid transport through capillary action (wicking)
    • Advantages
      • Wide range of applications
      • Inexpensive (~ $0.04 USD each)
      • Portable
      • Disposable
      • Simple operation
    Lu et al. 2009
  • 7. Assay Fabrication with Wax
    • Paper substrate
      • Whatman #1 filter paper
    • Designs drawn with graphics software
    • Printed with Xerox wax printer
      • 100 devices per sheet
    • Hot plate allows wax to melt through paper
    • Total fabrication time ~ 10 min
  • 8. Paper Assay for Bacteria
  • 9. Enzymes and their Substrates
  • 10. L. monocytogenes Assay: Characterization
    • Determine optimal substrate concentration for L. monocytogenes assay
    • Amount of PI-PLC enzyme is constant
    • Concentration of X-InP substrate varies
    • 80 mM is optimal
  • 11. L. monocytogenes Assay
    • Bacteria grown in TSB with yeast extract
    • Using 80 mM X-InP substrate
    • Performed assay at various enrichment time points
    • Able to detect L. monocytogenes within ~6 hr
    Control 2 hr 3 hr 3.5 hr 6 hr 5 hr 4.5 hr 4 hr
  • 12. Salmonella assay: pH Studies
    • Salmonella assay
      • Pure esterase enzyme
    • Used pH 7 in preliminary studies
    • Decided to use pH 9 for future work
  • 13. Salmonella Assay
    • Bacteria grown in TSB
    • Using 12 mM Magenta Caprylate substrate
    • Performed a pH study with live bacteria- some concern with using the pH 9 buffer
    • Assay performed after ~12 hr enrichment
    • Assay appears to work for wide range of pH values
    6.0 6.5 7.0 7.5 8.0 9.0 control
  • 14. E. coli Assay: pH Studies
    • E. coli assay
      • - Pure β -galactosidase enzyme
    • Decided to use pH 7.5 for future work
  • 15. E. coli Assay: Limit of detection
      • Determine lowest detectable amount of enzyme using 2.4 mM CPRG substrate
      • LOD for β -galactosidase: 0.03 ng/µL
    Varying β -galactosidase (µg/mL) control 0.03 0.06 0.13 0.4 0.33 0.26 0.2
  • 16. E. coli Assay
    • E. coli grown in 1 mL of growth media rather than 10 mL
    • More concentrated sample allows detection within 4.5 hr, decreasing assay time by ~ 2 hr
    Using 10 mL of growth media Using 1 mL of growth media for enrichment of E. coli (t = 4.5 hr) 7 mm
  • 17. Putting it all together: Detection of all the Bacteria
    • Goal: Detect all three bacteria types simultaneously
    • Substrates spotted in outer test zones
    • Solution containing all three enzymes in central sample well
    • Cross-reactivity tested for each assay
    E. coli Salmonella spp. control L. monocytogenes
  • 18. Future Work
    • Optimize size of paper devices
    • Continue conducting testing in foods
    • Cell phones
      • Image analysis
      • Quantification
  • 19. Thank you!

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