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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.

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

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  1. Novel Approaches & Probes for PCR Detection of Animal-Derived Materials Dr. Michael J. Myers US FDA, Center for Veterinary Medicine, Office of Research

  2. 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

  3. 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

  4. PCR Approach • Strengths • extremely sensitive • can detect single or multiple species (one method, multiple species) • Weakness • can’t distinguish between prohibited and exempt materials

  5. 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)

  6. 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)

  7. 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

  8. PCR Approach • Goals: • Develop PCR primer sets that are species specific • Species specific primers avoid the need for multiple assays for speciation

  9. “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)

  10. Universal Primer with Enzymatic Digestion cow deer elk sheep goat horse pig blank Intact Hinf 1 Hpy CH4 III

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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.”

  16. Acknowledgements • Dr. Haile F. Yancy • Dorothy E. Farrell

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