FDA Assessment of DNA BarCoding for FDA Assessment of DNA BarCoding for Species Identification of FishSpecies Identification of Fish

Part One: The Potential Use of DNA Part One: The Potential Use of DNA Barcodes in Regulatory Science Barcodes in Regulatory Science

Applications of the Regulatory Fish Applications of the Regulatory Fish EncyclopediaEncyclopedia

Haile Yancy, Ph. D. Haile Yancy, Ph. D.

U.S. FDA, Center for Veterinary U.S. FDA, Center for Veterinary Medicine, Office of ResearchMedicine, Office of Research

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

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

A DNA barcode is a short gene A DNA barcode is a short gene sequence used to identify sequence used to identify

species taken from a standard species taken from a standard position in the genomeposition in the genome

The Barcoding ProcessThe Barcoding Process

Extract DNA PCR COI Sequence COI

Specimen or Tissue Sample

DNA Barcode

Barcoding ProjectBarcoding Project Goals and ObjectivesGoals and Objectives

Develop proficient protocols to extract, Develop proficient protocols to extract, amplify, and sequence DNA to develop amplify, and sequence DNA to develop barcodesbarcodes

To create DNA barcodes of targeted To create DNA barcodes of targeted species of FDA’s interestspecies of FDA’s interest

Regulatory Fish Encyclopedia Regulatory Fish Encyclopedia (RFE)(RFE)

The Regulatory Fish Encyclopedia (RFE) is a The Regulatory Fish Encyclopedia (RFE) is a compilation of data in several formats that compilation of data in several formats that assists with the accurate identification of fish assists with the accurate identification of fish speciesspecies

It was developed by FDA's RFE Team to help federal, It was developed by FDA's RFE Team to help federal, state, and local officials and purchasers of seafood to state, and local officials and purchasers of seafood to identify species substitution and economic deception in identify species substitution and economic deception in the marketplacethe marketplace

http://www.cfsan.fda.govhttp://www.cfsan.fda.gov

Regulatory Fish Encyclopedia Regulatory Fish Encyclopedia (RFE)(RFE)

Includes 94 commercially relevant fish for sale in Includes 94 commercially relevant fish for sale in the U.S. marketthe U.S. market

– Visual comparisonVisual comparisonScanned digital images (whole fish and their market form)Scanned digital images (whole fish and their market form)

– Biochemical comparisonBiochemical comparisonIsoelectric focusing (IEF)Isoelectric focusing (IEF)

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

Problems with IEF IdentificationProblems with IEF Identification

Proteins Proteins – Sample conditionSample condition

Will not work on denatured samples Will not work on denatured samples (cooked/neglected)(cooked/neglected)

– Sample preparationSample preparationUses Association of Official Analytical Chemists Uses Association of Official Analytical Chemists (AOAC) validated method, but control samples are (AOAC) validated method, but control samples are becoming exhausted becoming exhausted

Different platformsDifferent platforms– Inconsistent gel patternsInconsistent gel patterns

CVM’s Proposed Supplement to CVM’s Proposed Supplement to the RFEthe RFE

To create barcodes for 94 fish contained in To create barcodes for 94 fish contained in the RFEthe RFE

Replace the restriction fragment length Replace the restriction fragment length polymorphism (RFLP) DNA information or polymorphism (RFLP) DNA information or to create barcode data linkto create barcode data link

FISH-BOLFISH-BOL

Dr. Paul Hebert and Dr. Dr. Paul Hebert and Dr. Bob Hanner Biodiversity Bob Hanner Biodiversity Institute of Ontario, Institute of Ontario, University of Guelph University of Guelph

Established June 2005Established June 2005

4,189 species barcoded4,189 species barcoded

21,981 barcodes21,981 barcodes

FISH-BOLFISH-BOL

FDA RFE projectFDA RFE project– 179 specimens179 specimens– 172 barcodes (sequences)172 barcodes (sequences)

73 species73 species

Blind Sample TestBlind Sample Test– 60 samples were tested60 samples were tested– 60/60 were identified correctly60/60 were identified correctly

Assay Development Using Assay Development Using Barcode DatabaseBarcode Database

PCR product ~650 bp

PCR product ~350-250 bp

PCR product ~250- 50 bp

Assay DevelopmentAssay Development

Microarray AssayMicroarray Assay– 40-80k species in single assay40-80k species in single assay– Ability to analyze cooked, processed, or mixed Ability to analyze cooked, processed, or mixed

samplessamples

Real- Time PCR AssayReal- Time PCR Assay– Develop assay in 2-3 daysDevelop assay in 2-3 days– 3-4 species in single assay3-4 species in single assay– Ability to analyze cooked, processed, or mixed Ability to analyze cooked, processed, or mixed

samplessamples– Results in ~1 hourResults in ~1 hour

Mixed Sample (tuna/catfish)

Tuna

TunaTuna

Ch. Ch. CatfisCatfishh

Blue Blue CatfisCatfishh

Allele IDOligo

………………………………………….......………………………………………..…………………………………………

Label

Fish Sample

………………………………………………………………………………………………………………………………………

Identify Species

(Tuna and Ch. Catfish)

Extract DNA

Create Zoo ChipHYB

Allele IDAllele ID

Allele ID aligns sequences of different species to identify Allele ID aligns sequences of different species to identify unique regionsunique regions

Species specific primers and probes are designed based Species specific primers and probes are designed based on differences in sequence on differences in sequence

Taxa specific primers and probes can also be designed Taxa specific primers and probes can also be designed

Designing Probes from Designing Probes from the COI Genethe 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’ 3’51 2 3 4

5Several Probes Designed Along Span of Gene 45-55 nucleotides in length

5’ 3’

Criteria for AnalyzingCriteria for Analyzing Microarray Slides Microarray Slides

Negative Negative (Spotting Solution Residues)

Positive

1. Color of spots (specificity)

2. Ratio of detection (red) to background (green) fluorescence above 2.0 (sensitivity)

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 ProbesTuna ProbeTuna Probe Ratio of red to green Ratio of red to green

fluorescencefluorescence

Probe 2Probe 2 14.6214.62

Probe 3Probe 3 11.8411.84

Probe 5Probe 5 12.3412.34

Fluorescence of Tuna DNA

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 1 5.34

Probe 5 20.34

Fluorescence of Rainbow Trout DNA

Real- Time PCR AssayReal- Time PCR Assay

Allele IDAllele ID

Aligned RFE and puffer fish barcode Aligned RFE and puffer fish barcode sequencessequences

Created three TaqmanCreated three Taqman®® probes that probes that detected all species of puffer fish contained detected all species of puffer fish contained in the BOLD databasein the BOLD database– PU016PU016– PU050PU050– PU038PU038

Pufferfish Assay MethodsPufferfish Assay Methods

Extract DNAExtract DNA

TaqmanTaqman®® RT-PCR Protocol RT-PCR Protocol– 95°C: 2 minutes95°C: 2 minutes– 50 cycles50 cycles

95°C 15 sec. Denaturation95°C 15 sec. Denaturation

60°C 30 sec. Annealing and Extension60°C 30 sec. Annealing and Extension

Analyze ResultsAnalyze Results

Optimization of Pufferfish PrimersOptimization of Pufferfish Primers

Pufferfish Primer PU016: Ct=25.44

Pufferfish Primer PU050: Ct=25.5

Pufferfish Primer PU038: Ct=42.7

Pufferfish Primer PU016 SpecificityPufferfish 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

Known Pufferfish Samples with Known Pufferfish Samples with Primer PU016Primer PU016

Left: Florida Southern Puffer: Ct=22.84

Right: Florida Bandtail Puffer: Ct=30.7

Left: Florida Checkered Puffer: Ct=41.54

Cooked Fish from SoupCooked Fish from Soup

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

Pufferfish Assay Pufferfish Assay

PU016PU016PU050PU050PU038PU038

Next Step of Assay ValidationNext Step of Assay Validation

FDA is in the process of submitting the FDA is in the process of submitting the assay for AOAC® assay for AOAC® Peer-Verified MethodPeer-Verified Method

After validation, the pufferfish assay will be After validation, the pufferfish assay will be used by Office Of Regulatory Affairs used by Office Of Regulatory Affairs (ORA) labs(ORA) labs

ARegulatory Tool

for theThird

Millennium

AcknowledgementsAcknowledgementsCVMCVM

Dr. Marleen WekellDr. Marleen WekellDr. Russell FrobishDr. Russell FrobishJewell WashingtonJewell WashingtonJacquline MasonJacquline MasonMichelle LeCluyseMichelle LeCluyse

CFSANCFSANDr. Fred FryDr. Fred FrySpring RandolphSpring RandolphDr. Jon DeedsDr. Jon Deeds

CONSORTIUM FOR THE CONSORTIUM FOR THE BARCODE OF LIFEBARCODE OF LIFE

Dr. Paul HebertDr. Paul HebertTyler ZemlakTyler ZemlakDr. Scott MillerDr. Scott MillerDr. David SchindelDr. David SchindelDr. Bob HannerDr. Bob Hanner

Seafood Products Research Center Pacific Seafood Products Research Center Pacific Regional Laboratory – NorthwestRegional Laboratory – Northwest

Dr. Rick LongDr. Cindy WuJim BarnettNgoc-Lan Nguyen Dr. Michelle MooreDr. Bradley Tenge

Thank You Thank You

Questions?Questions?