Lawrence Goodridge

Department of Animal Sciences

Colorado State University

Lawrence.Goodridge@Colostate.edu

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 AssaysPreviously Developed

Salmonella Listeria monocytogenes

Escherichia coli

Disadvantages of theBiochemical 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

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

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 hr5 hr4.5 hr4 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

7 mm

Using 10 mL of growth media Using 1 mL of growth media for enrichment of E. coli (t = 4.5 hr)

Putting it all together: Detection of all the Bacteria

E. coli

Salmonella spp.

control

L. monocytogenes

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

Future Work

• Optimize size of paper devices• Continue conducting testing in

foods

• Cell phones– Image analysis – Quantification

Thank you!