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Determination of Animal Species by PCR in
Feed Lars Reimann and David Piñero Eurofins
Scientific, Inc. January 17, 2003
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Eurofins Scientific Who Are We?

• Leading global bioanalytical services provider
  for
• Feed
• Food
• Pharma/Biotechnology
• Cosmetics
• Environmental
• Broad portfolio of 3000 methods and proprietary
  assays
• gt 2.000 employees
• More than 50 laboratories in Europe and North
  America

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USA Presence

• Woodson-Tenent Laboratories Division
• Feed and Feed Ingredients
• Food and Food Ingredients
• Biotechnology testing (GMO, Meat Speciation)
• Authenticity (Adulteration, Traceability)
• Alpha Laboratories Division
• Herbal products
• Food Supplements

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Contents of Presentation

• PCR and Real-Time PCR Technology
• PCR Advantages/Limitations
• Analysis Procedure
• Validation
• Quality Control/Quality Assurance
• Conclusion

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Why Concerned About Meat Speciation

• Meeting Regulatory Restrictions (FDA, EU, Japan,
  China)
• BSE/Scrapies and other Food Safety Concerns
• Religious Concerns
• Adulteration of High Value Products

• Brand Protection Labelling Issues

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Meat Speciation MethodologiesPCR (Principle and
Types)

• Regular PCR (2 species specific primers)
• RFLP PCR (Restriction Fragment Length
  Polymorphism)
• Immuno-PCR (PCR-ELISA)
• Semi-Quantitative PCR
• Real-Time PCR

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PCR (Advantages)

• High species specificity
• High sensitivity. Detection limits 0.1 of DNA
  present.
• Semi-quantitative PCR can provide a rough
  estimate of the level of contamination
• Real-Time PCR providing quite accurate
  quantitation in the less than 10 range
• DNA highly stable

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PCR (Limitations)

• Currently no tissue specificity
• Not all tissues contain the same amount of DNA
• Require relatively advanced lab facilities and
  instrumentation and highly trained staff
• Cost is fairly expensive (75- 300)
• Time (usually gt24 hrs for results)
• Special measures needed for avoidance of
  contamination and inhibition

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PCR

• Detects a known DNA sequence by amplification
• Target sequence is duplicated billions of times
• Results obtained can be quantitative or
  qualitative
• Amplification is done in changing temperature
  cycles
• Enzyme Thermophyllic TAQ polymerase
• Instrument Thermocycler

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Usual Target Sequences

• Species-specific nuclear satellite DNA (i.e.
  Satellites, SINEs, etc.)
• Mitochondrial polymorphic multi-copy genes (i.e.
  cytB, rDNA)

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Steps in a PCR Cycle

• Denaturation (90-95 º C)
• Primer Annealing (40-70 º C)
• Extension (72 º C)

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Polymerase Chain Reaction (PCR)
PRIMER ANNEALING
POLYMERASE RECOGNITION
THE STRAND IS DUPLICATED
DENATURATION
MAGNESIUM COFACTOR
EXTENSION
TAAACTAGCGAAACT
GCGTAGTGTGTCTAATCCGTGGACGACTTATT
ATTTGATCGCTTTGACGCATCACACAGATTAGGCACCTGCTGAATAA
TAAACTAGCGAAACTGCGTAGTGTGTCTAATCCGTGGACGACTTATT
CACCTGCTGAATAA
ATTTGATCGCTTTGACGCATCACACAGATTAGG
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Theoretical DNA Amplification
Cycle of DNA copies 1 2 2 4 4 16 10
1,024 15 32,768 20 1,048,576 25 33,554,432
30 1,073,741,824
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Real-Time PCR
Source Boehringer Mannheim Perkin Elmer
Applied Biosystems WWW page
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Denaturation
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Annealing
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Extension
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5 Exonuclease Activity
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(No Transcript)
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Analysis Procedure

• Sample Preparation
• DNA Extraction
• PCR Preparation
• PCR
• Post-PCR

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Agarose Gel Electrophoresis
UV Photograph Polaroid
Photograph
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Validation

• Sensitivity
• Specificity
• Primer systems tested against other species
• Primer systems tested against DNA dilutions in
  various percentages and/or samples with different
  weight-to weight ratios
• Accuracy/Precision
• Primer systems tested in replicates against
  various matrices of known composition
• Robustness
• European sister labs able to duplicate results

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Example Goat Real-Time PCR Sensitivity
.1

• Minimum detection limit 0.05 goat DNA

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Sensitivity of Current Procedures

• Bovine and Caprine Real-Time PCR Sensitivity
  0.05
• Poultry, Porcine, Ovine and Caprine standard PCR
  sensitivities 0.1
• Bovine standard PCR sensitivity 0.125
• All primer systems tested specific under our
  method specifications

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PCR Quality Control/Quality Assurance

• Positive and negative controls
• Extraction controls (Blanks)
• Control reactions (i.e. cytB gene), spikes, IPCs
• Validated primer systems
• All analysis performed at least in duplicate
• Participation in sponsored check sample programs/
  collaborative studies
• Internal validation trials

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Conclusion

• PCR and Real-Time PCR have proven to be rugged
  methods for determining the species represented
  in animal protein products
• No single test conclusively addresses the issue
  of regulatory compliance
• Theoretically, no current battery of tests can
  conclusively confirm failure to comply with
  current regulations

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Thank you
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Contact us

• Lars Reimann
• Telephone [phone] Ext. 112
• Fax [phone]
• Email larsreimann_at_eurofins.com
• David Pinero
• Telephone [phone]
• Fax [phone]
• E-mail davidpinero_at_eurofins.com