Integrating Detection of Multiple Pathogens in Food

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Integrating Detection of Multiple Pathogens in Food

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

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