Integrating Detection of Multiple Pathogens in Food

Lawrence Goodridge Department of Animal Sciences Colorado State University [email_address] Integrating Detection of Multiple Pathogens in Food

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

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

Biochemical Assays Previously Developed Salmonella Listeria monocytogenes Escherichia coli

Disadvantages of the Biochemical Assays
Still too labor intensive
Sensitivity and substrate issues
Can only detect one microorganism at a time

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

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

Paper Assay for Bacteria

Enzymes and their Substrates

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

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

Salmonella assay: pH Studies
Salmonella assay
Pure esterase enzyme
Used pH 7 in preliminary studies
Decided to use pH 9 for future work

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

E. coli Assay: pH Studies
E. coli assay
- Pure β -galactosidase enzyme
Decided to use pH 7.5 for future work

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

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

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

Future Work
Optimize size of paper devices
Continue conducting testing in foods
Cell phones
Image analysis
Quantification

Thank you!

Integrating Detection of Multiple Pathogens in Food

by dedmark on Sep 19, 2011

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Integrating Detection of Multiple Pathogens in Food — Presentation Transcript