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Novel Approaches & Probes for PCR Detection of Animal-Derived Materials. Dr. Michael J. Myers US FDA, Center for Veterinary Medicine, Office of Research. Reasons for FDA’s Concern with Animal Feed.

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Novel approaches probes for pcr detection of animal derived materials l.jpg

Novel Approaches & Probes for PCR Detection of Animal-Derived Materials

Dr. Michael J. Myers

US FDA, Center for Veterinary Medicine, Office of Research


Reasons for fda s concern with animal feed l.jpg
Reasons for FDA’s Concern with Animal Feed Animal-Derived Materials

  • Epidemiological evidence associated BSE with animal feed containing rendered material derived from scrapie infected sheep

  • Epidemiological data associating BSE with vCJD


Scope of problem for cvm 1997 feed ban l.jpg
Scope of Problem for CVM: Animal-Derived Materials1997 Feed Ban

  • Species Specificity

    • Cattle, sheep, goats, deer, and elk are prohibited

    • Pure equine and porcine exempt

  • Tissue specificity

    • meat and bone meal prohibited: plate waste exempt

    • Gelatin, milk and blood meal exempt tissues


Pcr approach l.jpg
PCR Approach Animal-Derived Materials

  • Strengths

    • extremely sensitive

    • can detect single or multiple species (one method, multiple species)

  • Weakness

    • can’t distinguish between prohibited and exempt materials


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Current PCR Method Animal-Derived Materials

  • One sample can be analyzed in under 24 hr

    • Solubilize 0.5 g feed (~16 hr)

    • Extract DNA

    • Perform PCR

    • Gel electrophoresis to detect results

      (Tartaglia et al JFP 61:513-518)


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Results from Validation of Bovine PCR Primers Animal-Derived Materials

  • Rate of false positive was 0.83%

  • Overall rate of false negatives was 1.25%

    • at 2% BMBM level: 0.83%

    • at 0.125% BMBM level: 1.67%

      (Myers et al., 2001, JFP 64:564-566)


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Current CVM BSE Initiatives Animal-Derived Materials

  • Detection of multiple species by PCR

    • deer, elk, sheep, goat, pig (primary interest)

    • dog, cat, horse, chicken, goose, and turkey DNA’s used to help validate specificity

  • Development of a new ELISA test

    • Capable of distinguishing prohibited from exempt bovine materials


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PCR Approach Animal-Derived Materials

  • Goals:

    • Develop PCR primer sets that are species specific

    • Species specific primers avoid the need for multiple assays for speciation


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Animal-Derived MaterialsUniversal” PCR Primer Pair

sheep

elk

dog

horse

goat

cow

deer

pig

turkey

cat

rabbit

dog

chicken

goose

deer

pig

(Myers et al., 2003, JFP accepted)


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Universal Primer with Enzymatic Digestion Animal-Derived Materials

cow

deer

elk

sheep

goat

horse

pig

blank

Intact

Hinf 1

Hpy CH4 III


Application of pcr dog food animal feed with universal primer l.jpg
Application of PCR: Animal-Derived MaterialsDog Food & Animal Feed with Universal Primer

Dog food

Dairy Feed

MW

Chicken

& Fish

Pig Blood

meal

Bovine

MBM

Pig Blood

Meal

Lamb

Chicken

Turkey

Blank

Intact

Hinf 1


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PCR Analysis of Pig Blood Meal with Enzyme Cutting Animal-Derived Materials

Bovine Primers

Porcine Primers

Universal Primers

Blank

Pig Blood

meal

Dairy Feed

With PBM

Pig Blood

meal

Dairy Feed

With PBM

Pig Blood

meal

Dairy Feed

With PBM


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PCR Method: Summary Animal-Derived Materials

  • Identified a PCR primer pair that detects most prohibited species

    • Detects cow, sheep, goat, deer, elk

    • Detects horse and swine, which are exempt

    • Enzyme cutting can identify pig or horse

  • Identified species-specific PCR primers for:

    • cattle, sheep/goat, pig, horse, poultry


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PCR Approach: Conclusion Animal-Derived Materials

  • Development of a PCR-based approach to determine species identity in feed and feed ingredients

  • Test to detect adulteration or misbranding of feed and feed ingredients


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Future Directions Animal-Derived Materials

  • Method Validation trial

    • Using several different PCR primers and feed ingredients & a shorter DNA extraction step.

    • Currently have 7 labs committed

    • Need 1 more (minimum)

  • Assessment of carry-over

    • What amount of prohibited material is found in feed following “best-industry practices.”


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Acknowledgements Animal-Derived Materials

  • Dr. Haile F. Yancy

  • Dorothy E. Farrell


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