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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

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
Reasons for FDA’s Concern with Animal Feed
  • 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
Scope of Problem for CVM:1997 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
PCR Approach
  • Strengths
    • extremely sensitive
    • can detect single or multiple species (one method, multiple species)
  • Weakness
    • can’t distinguish between prohibited and exempt materials
current pcr method
Current PCR Method
  • 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)

results from validation of bovine pcr primers
Results from Validation of Bovine PCR Primers
  • 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)

current cvm bse initiatives
Current CVM BSE Initiatives
  • 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
pcr approach8
PCR Approach
  • Goals:
    • Develop PCR primer sets that are species specific
    • Species specific primers avoid the need for multiple assays for speciation
universal pcr primer pair
“Universal” 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)

universal primer with enzymatic digestion
Universal Primer with Enzymatic Digestion

cow

deer

elk

sheep

goat

horse

pig

blank

Intact

Hinf 1

Hpy CH4 III

application of pcr dog food animal feed with universal primer
Application of PCR: Dog 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

pcr analysis of pig blood meal with enzyme cutting
PCR Analysis of Pig Blood Meal with Enzyme Cutting

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

pcr method summary
PCR Method: Summary
  • 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
pcr approach conclusion
PCR Approach: Conclusion
  • 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
future directions
Future Directions
  • 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.”
acknowledgements
Acknowledgements
  • Dr. Haile F. Yancy
  • Dorothy E. Farrell
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