1. FDA Assessment of DNA BarCoding for Species Identification of Fish Part One: The Potential Use of DNA Barcodes in Regulatory Science Applications of the Regulatory Fish Encyclopedia Haile Yancy, Ph. D. U.S. FDA, Center for Veterinary Medicine, Office of Research

2. The U.S. Food and Drug Administration (FDA) is responsible for protecting the public health by assuring the safety, efficacy, and security of food, human and veterinary drugs, biological products, medical devices, cosmetics, and products that emit radiation.

3. Center for Veterinary Medicine (CVM) is responsible for regulating, drugs, devices, feeds and feed additives given to over one hundred million companion animals, plus millions of poultry, cattle, swine, and minor animal species.

4. A DNA barcode is a short gene sequence used to identify species taken from a standard position in the genome

5. The Barcoding Process Extract DNA PCR COISequence COI Specimen or Tissue Sample DNA Barcode

6. Barcoding Project Goals and Objectives Develop proficient protocols to extract, amplify, and sequence DNA to develop barcodes To create DNA barcodes of targeted species of FDA’s interest

7. Regulatory Fish Encyclopedia (RFE) The Regulatory Fish Encyclopedia (RFE) is a compilation of data in several formats that assists with the accurate identification of fish species It was developed by FDA's RFE Team to help federal, state, and local officials and purchasers of seafood to identify species substitution and economic deception in the marketplace http://www.cfsan.fda.gov

8. Regulatory Fish Encyclopedia (RFE) Includes 94 commercially relevant fish for sale in the U.S. market –Visual comparison Scanned digital images (whole fish and their market form) –Biochemical comparison Isoelectric focusing (IEF)

10. Isoelectric Focusing (IEF) Protein Fingerprint Isoelectric focusing protein patterns for the pH range 3-10 for fresh, frozen, uncooked seafood based on AOAC 980.16

11. Problems with IEF Identification Proteins –Sample condition Will not work on denatured samples (cooked/neglected) –Sample preparation Uses Association of Official Analytical Chemists (AOAC) validated method, but control samples are becoming exhausted Different platforms –Inconsistent gel patterns

12. CVM’s Proposed Supplement to the RFE To create barcodes for 94 fish contained in the RFE Replace the restriction fragment length polymorphism (RFLP) DNA information or to create barcode data link

13. FISH-BOL Dr. Paul Hebert and Dr. Bob Hanner Biodiversity Institute of Ontario, University of Guelph Established June 2005 4,189 species barcoded 21,981 barcodes

14. FISH-BOL FDA RFE project –179 specimens –172 barcodes (sequences) 73 species Blind Sample Test –60 samples were tested –60/60 were identified correctly

16. Assay Development Using Barcode Database

17. PCR product ~650 bp PCR product ~350-250 bp PCR product ~250- 50 bp

18. Assay Development Microarray Assay –40-80k species in single assay –Ability to analyze cooked, processed, or mixed samples Real- Time PCR Assay –Develop assay in 2-3 days –3-4 species in single assay –Ability to analyze cooked, processed, or mixed samples –Results in ~1 hour

19. Mixed Sample (tuna/catfish) Tuna

20. Tuna Ch. Catfish Blue Catfish Allele ID Oligo ………………………………………….......………………………………………..………………………………………… Label Fish Sample …………………………………………… …………………………………………… …………………………………………… Identify Species (Tuna and Ch. Catfish) Extract DNA Create Zoo Chip HYB

21. Allele ID Allele ID aligns sequences of different species to identify unique regions Species specific primers and probes are designed based on differences in sequence Taxa specific primers and probes can also be designed

22. Designing Probes from the COI Gene 5’3’ Generally probes are designed at a specific place along a gene sequence 5’3’ Available DNA of cooked, processed, or degraded sample 5’3’ 5 1234 5 Several Probes Designed Along Span of Gene 45-55 nucleotides in length 5’3’

23. Criteria for Analyzing Microarray Slides NegativeNegative (Spotting Solution Residues) Positive 1. Color of spots (specificity) 2. Ratio of detection (red) to background (green) fluorescence above 2.0 (sensitivity)

24. Ch. Catfish DNA with Tuna Probes Blue Catfish DNA with Tuna Probes Rainbow Trout DNA with Tuna Probes Great Barracuda DNA with Tuna Probes Microarray results: Binding of DNA to Tuna Probes Tuna DNA with Tuna Probes Tuna Probe Ratio of red to green fluorescence Probe 2 14.62 Probe 3 11.84 Probe 5 12.34 Fluorescence of Tuna DNA

25. Great Barracuda DNA with Rainbow Trout probes Rainbow Trout DNA with Rainbow Trout probes Blue Catfish DNA with Rainbow Trout probes Channel Catfish DNA with Rainbow Trout probes Binding of DNA to Rainbow Trout Probes Rainbow Trout Probe Ratio of red to green fluorescence Probe 15.34 Probe 520.34 Fluorescence of Rainbow Trout DNA

26. Real- Time PCR Assay Allele ID Aligned RFE and puffer fish barcode sequences Created three Taqman ® probes that detected all species of puffer fish contained in the BOLD database –PU016 –PU050 –PU038

27. Pufferfish Assay Methods Extract DNA Taqman ® RT-PCR Protocol –95°C: 2 minutes –50 cycles 95°C 15 sec. Denaturation 60°C 30 sec. Annealing and Extension Analyze Results

28. Optimization of Pufferfish Primers Pufferfish Primer PU016: Ct=25.44 Pufferfish Primer PU050: Ct=25.5 Pufferfish Primer PU038: Ct=42.7

29. Pufferfish Primer PU016 Specificity Figure 1. Primer PU016 was tested against 16 other fish species closely related to pufferfish to determine specificity. All samples came up negative

30. Known Pufferfish Samples with Primer PU016 Left: Florida Southern Puffer: Ct=22.84 Right: Florida Bandtail Puffer: Ct=30.7 Left: Florida Checkered Puffer: Ct=41.54

31. Cooked Fish from Soup Figure 1. Cooked puffer sample from the soup that caused the illnesses. Primer PU016 was used. Ct=41.26

32. Pufferfish Assay PU016PU050PU038

33. Next Step of Assay Validation FDA is in the process of submitting the assay for AOAC® Peer-Verified Method After validation, the pufferfish assay will be used by Office Of Regulatory Affairs (ORA) labs

34. A Regulatory Tool for the Third Millennium

35. Acknowledgements CVM Dr. Marleen Wekell Dr. Russell Frobish Jewell Washington Jacquline Mason Michelle LeCluyse CFSAN Dr. Fred Fry Spring Randolph Dr. Jon Deeds CONSORTIUM FOR THE BARCODE OF LIFE Dr. Paul Hebert Tyler Zemlak Dr. Scott Miller Dr. David Schindel Dr. Bob Hanner Seafood Products Research Center Pacific Regional Laboratory – Northwest Dr. Rick Long Dr. Cindy Wu Jim Barnett Ngoc-Lan Nguyen Dr. Michelle Moore Dr. Bradley Tenge

36. Thank You Questions?