Detection and enumeration of Listeria monocytogenes in foods : - - PowerPoint PPT Presentation

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Detection and enumeration of Listeria monocytogenes in foods : assets and limits

Mansel W. Griffiths University of Guelph

What do we need to detect?

• Listeria spp.

– Environmental monitoring – USDA regulations

• Listeria monocytogenes

– Foods

• Listeria monocytogenes – human pathogens

– Foods – Clinical samples

What test attributes do we need?

• Sensitive and specific
• Speed
• Precision and accuracy
• Easy-to-use
• Cost effective
• Approved by regulatory agencies

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Culture–based

Environmental testing

• Paradigm PDX 24 - The

24 Hour Test for Listeria in Environmental Samples

• Esculin hydrolysis
• Swab based

Chromogenic agar - Oxoid

• ∃-glucoside chromogen

cleaved by ∃-glucosidase, common to all Listeria

• blue/green colonies
• Phospholipase in L.

monocytogenes (and pathogenic L. ivanovii) hydrolyses lecithin producing distinctive

• paque white halo

O.B.I.S. (Oxoid Biochemical Identification Systems)

• All Listeria species except L.

monocytogenes, possess D- alanyl aminopeptidase (DALAase) activity.

• O.B.I.S. mono detects this

enzyme using D-alanyl-7-amido- 4- methylcoumarin (DALA)

• Colour appears in 20 secs and

indicates that the suspect colonies are NOT L. monocytogenes

Biosys

• Monitors changes in

chemical characteristics

• f liquid medium
• Reagents change

spectral patterns as metabolic process takes place

• Changes are detected

photometrically at predetermined time intervals in a semifluid zone separated from the liquid medium

Sample preparation Pathatrix

• Listeria
• Listeria &

Salmonella

–Processes large volumes

What is currently available? Immunoassays

• Automated

– BioMerieux Vitek

• Vidas
• Mini Vidas

– TECRA

• Unique

What is currently available? Immunoassays

• Lateral flow

– Biocontrol

• VIP for Listeria

– Neogen

• Reveal for Listeria

The future of immunoassays?

• Immunoassays

– Immobilize Ab on plastic film

• Toxin Alert

– Cheap source of Ab

• Plantibodies

– Patented in-situ immunoassay

In situ Immunoassays:

Real-time detection in foods

Organism present Organism absent

Genomics-based assays Real-Time PCR

• A new generation of PCR instruments and reagents

allows simultaneous amplification and quantification

• f specific nucleic acid sequences.
• Detect the DNA generated within a PCR on a cycle-

by-cycle basis.

• Provides greater quantitative precision and greater

convenience.

• Fluorescence-detecting thermocyclers.

Detection SYBR-Green Assays

• Intercalating dye
• Multiplex assays for

Salmonella and L. monocytogenes

• Results within 1.5 h

BAX System

• 1. Samples enriched and treated to

release DNA.

• 2. PCR tablets, containing all

reagents necessary for PCR hydrated with lysed sample. Processed in cycler/detector to amplify a DNA fragment specific to the target.

• 3. The amplified DNA generates a

fluorescent signal. Results displayed as +ve or -ve symbols

BAX-System

• Accurate

– based on DNA – simplified sample preparation procedure – automated detection

• Rapid

– next day results – 96 samples within 4 h after sample prep. – almost 200 tests per day,

• Convenient operation and

more control

– standard laboratory techniques – tableted reagents – closed-tube system – simple on-screen reporting

• Approved

– Health Canada; FSIS; AOAC

GeneVision (Warnex Inc.)

• Detection of L.

monocytogenes based

• n real-time PCR
• Molecular beacons
• Approved by Health

Canada

• Adopted by Plumrose,

Cardinal Meats

Target Probe Hybridization

1 cm2 = 25,000 probes spotted

DNA Microarrays

Evaluation: specificity testing

• C. parvum
• G. intestinalis
• C. cayetanensis
• E. coli

STEC 1&2

• S. typhimurium

DT104

• E. coli

STEC 1&2 O157:H7 Salmonella

• C. coli
• L. mono.

Key

Dye Buffer 16S

The Future

• Better methods to concentrate and remove

Listeria from foods

• Genetic based sensors to discriminate

between pathogenic strains

• Micro-fluidics devices