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Advances in Molecular Techniques
for Food Safety
George TiceDirector, Research and Development
DuPont Qualicon
5/27/2010
2
Food testing – process flow
Collect
samples
EnrichPrepare
samplesDetect
target
Isolate &
Characterize
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3
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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12
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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