Advances in Molecular Techniques

for Food Safety

George TiceDirector, Research and Development

DuPont Qualicon

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Food testing – process flow

Collect

samples

EnrichPrepare

samplesDetect

target

Isolate &

Characterize

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Discovery of DNADiscovery of DNA

1953 J. Watson, USA, and F. Crick, UK, show that the DNA molecul1953 J. Watson, USA, and F. Crick, UK, show that the DNA molecule consists of a double helix, thus e consists of a double helix, thus

making one of the most important discoveries of this centurymaking one of the most important discoveries of this century

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DNA Structure

Deoxyribonucleic acid:

A linear polymer that consists of four nucleotides:

Adenine

Cytosine

Guanine

Thymine

Primer binding

A - T C - G

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Primers are specific to DNA fragment

3’AAAAAAAATGCATGCATTTCCCAAAAAAAAAA5’

5’ACGTACGTAAAGGG3’

||||||||||||||

primer

template

5’ACGTACGTAAAGGG3’||||||||

XXXXXXXXXX

template

primer

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Amplification techniques

Nucleic Acid Sequence based Amplification (NASBA)

Transcription Mediated Amplification (TMA)

Strand Displacement Reaction (SDA)

Ligase Chain Reaction (LCR)

Reverse Transcriptase

Polymerase Chain Reaction (PCR)

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Polymerase

Chain

Reaction

(PCR)

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PCR amplifies specific fragments only

+

+ no detectable

amplification

Target organism DNAPrimer specific

amplification PCR

Background DNA

PCR

Primer specific

amplification

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PCR gel detection

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PCR automated detection- melting curve analysis

No SignalSignal

Increasing temperature

Fluorescent dye

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Algorithms convert raw melt curve to processed peaks

Raw

Data

Processed

Data

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D Q

F

Light source

Fluorescent Resonate Energy Transfer (FRET)

Q

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Scorpion Primer

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Amplification plots

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Quantitative PCRQuantitative PCR

Cycle number

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Advantages of Amplification Techniques

• Detects specific microbial DNA sequences within a background of high levels of non-target DNA.

• Rapidly produces sufficient copies of specific DNA for easy detection

• More sensitive than conventional techniques

• Highly specific

• Does not rely on phenotypic expression of antigens

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Disadvantages of Amplification Techniques

•The process requires instrumentation

•Sample preparation

•Amplicon contamination of work environment

•PCR inhibitors from the sample matrix

•Culture confirmation could be difficult

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Current applications

Pathogen detection

• Salmonella

• E. coli O157:H7

• Listeria monocytogenes

• Campylobacter

• Cronobacter spp

• Vibrio

Indicator tests

• Listeria spp

• Entereobacteriaceae

Quality tests

• Y&M

• Staph aureus

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Food testing – future process – integrated

Collect

samples

EnrichPrepare

samplesDetect

target

Isolate &

Characterize

Food and

Env. Samples

Pooling

Advanced

materials

Enrich during

transit

Media to enhance

bacterial growth

Simplified broths

Selective broths

Wet pooling

Development

Focus

Concentration

Matrix clean-up

BAX® System real-time assays

Microfluidics

System integration

Selected media

DNA Fingerprinting

Improved selective

agars

Faster, more robust

char. systems

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