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  • Agilent Technologies

    Fish Species

    Identification System

    AOAC Annual Meeting

    Exhibitor Presentation

    Scott Happe, Ph.D.

    Senior Research Scientist

    Stratagene Products Division

    scott.happe@agilent.com

    September 15, 20091

  • Preventing Fraud, Mislabeling, and Substitution:

    Primary Drivers for Fish Identification Testing

    September 15, 20092

    Consumer Satisfaction/

    Product Value

    Sustainability and

    Environmental Protection

    Regulatory Compliance

    Safety

  • Fish Identification Methods

    • Requires expert for identification

    • Only applicable to whole fish

    Morphology

    • Processing can destroy proteins

    • Limited polymorphism between species

    • Difficult to interpret (e.g. IEF)

    • Influenced by environment

    Protein-based methods

    • Objective results

    • More resistant to sample processing

    • Easy to perform

    • High specificity/sensitivity

    DNA-based methods

    Isoelectric focusing

    ELISA

    PCR/QPCR

    RFLP

    Sequencing

    September 15, 20093

  • SPECIES 1

    Polymerase Chain Reaction (PCR) –

    Restriction Fragment Length Polymorphism (PCR-RFLP)

    D ig

    e s

    t w

    it h

    E n

    z y m

    e B

    SPECIES 2

    E L

    E C

    T R

    O P

    H O

    R E

    S IS

    PCR:

    Amplify with generic

    fish primers

    Sample DNA

    PCR product:

    “amplicon”

    September 15, 20094

  • Basis for the Fish Species Identification System is

    the PCR-RFLP Method Developed at Campden BRI

    September 15, 20095

    Food Control 16 (7), 601-607

    http://www.chem.agilent.com/Library/applications/5989-2982EN.pdf

  • Agilent Bioanalyzer 2100

    September 15, 20096

    • First commercially available

    Lab-on-a-Chip product (1999)

    • Industry‘s ONLY microfluidics-

    based platform that can

    analyze DNA, RNA, proteins

    and cells

    • Results in ~30 minutes

    • More than 6500 publications

    • Gold-Standard for the analysis

    of RNA

    • 21 CFR Part 11 compliant

    (electronic records)

  • Chip and Electrode Cartridge Molecular Assays

    On-chip gel electrophoresis for the analysis of RNA, DNA and proteins

    • Chips accommodate 12 sample wells, gel wells

    and a well for a sizing standard (ladder)

    • Gel matrix is injected into etched channels

    on the chip

    • Sample mixed with size marker and loaded

    • A16-pin electrode in the lid fits into the wells

    on the chip

    • Electrokinetic force drives DNA through the

    separation channel and past the detector

    Gel wells

    Ladder well Separation channel and point of detection

    7 September 15, 2009

    Disposable

    DNA chip

  • Bioanalyzer Instrumentation Enhances

    Ease-of-Use and Accuracy of Pattern Matching

    • Visual inspection of agarose gels and comparison to validated patterns is tedious and prone to error

    • Bioanalyzer fragment resolution is superior to agarose gels

    • Peak information can be automatically extracted and fed into pattern

    matching software

    September 15, 20098

    Agarose Gel Readout

    Bioanalyzer Readout

  • Extraction PCR RFLP

    Fish Identification

    Agilent Fish Species ID Platform

    Agilent 2100

    Bioanalyzer

    RFLP Matcher

    Software

    September 15, 20099

    ~6 hrs total time

    from sample to

    results

    < 1 hr ~ 1.5-2 hrs 2.25 hrs

  • 1. Measure 40 mg – 1 g tissue

    (fresh, frozen, or processed)

    Homogenization optional

    2. Digest with Proteinase K Digestion Buffer

    Incubate at 65oC for 10 min

    3. Capture DNA onto glass-fiber spin column

    4. Wash with High Salt Buffer (1X)

    Wash with 80% Ethanol (3X)

    5. Elute DNA

    100 µl of Elution Buffer

    Step 1: Extraction of DNA from Fish Tissue Using the

    Fish Species Identification System

    < 1 hr from

    tissue to DNA

    Page 10

  • High DNA Yield from A Variety of Fish Samples

    Page 11

    0

    2

    4

    6

    8

    10

    12

    14

    16

    18

    Atlantic

    Salmon -

    wild

    Tilapia-

    wild

    Catfish -

    farm

    Pacific

    Cod -

    wild

    Tilapia-

    farm

    Scallop -

    wild

    Trout

    Reinbow

    - farm

    Dover -

    wild

    Tuna -

    wild

    Salmon -

    farm

    Basa

    Swai -

    farm

    Snapper

    Pacific-

    wild

    Yield range: 2-18 µg from 40 mg tissue

    Purity: Average of 2.19 A260/A280

    PCR input requirement: 0.5 – 500 ng T o

    ta l y ie

    ld , µ

    g

    T o

    ta l y ie

    ld , µ

    g

    0

    2

    4

    6

    8

    10

    12

    14

    16

    1 2 3 4 5 6 7 8 9 10 11 12

    Competitor Kits Agilent Kit

    Average

    260/280: 1.41 Average

    260/280: 2.09

    September 15, 2009

    Extractions from a Variety of Fresh Fish

    0

    5

    10

    15

    20

    25

    30

    35

    40

    45

    50

    Dry fish Canned fish Fish fins

    Yield from Alternative Fish Samples

    (40 mg each)

    D N

    A Y

    ie ld

    , u

    g

    6.6µg 3.7µg

    43µg

  • PCR Target: 464 bp Region of the Fish cyt b Locus Polymorphic region flanked by conserved sequence

    September 15, 200912

  • Step 2: PCR

    cytb

    PCR Amplicon

    PCR Master Mix

    Fish Primer Mix

    + 1 µl Fish DNA Extract OR

    Positive Control Salmon DNA

    PCR Cycling

    1.5-2 hrs

    September 15, 200913

    http://www.stratagene.com/products/showCategory.aspx?catId=25 http://rds.yahoo.com/_ylt=A9G_bDnZJZxKsh0BjKWjzbkF/SIG=12ns4cm1p/EXP=1251833689/**http:/media.biocompare.com/bcimages/spotlight/sp367_stratagene.jpg http://rds.yahoo.com/_ylt=A9G_bDnZJZxKsh0BjKWjzbkF/SIG=12ns4cm1p/EXP=1251833689/**http:/media.biocompare.com/bcimages/spotlight/sp367_stratagene.jpg

  • P a

    c if

    ic C

    o d

    D o

    v e

    r

    T il

    a p

    ia (

    fa rm

    )

    s c

    a ll

    o p

    S n

    a p

    p e

    r

    T ro

    u t

    T u

    n a

    A tl

    a n

    ti c

    s a

    lm o

    n

    S w

    a i

    C a

    tf is

    h

    S a

    lm o

    n (

    fa rm

    )

    T il

    a p

    ia (

    w il d

    )

    Validation of Amplification from Different Fish Samples

    September 15, 200914

  • Step 3: Restriction Digestion

    D ig

    e s

    t w

    it h

    H a

    e II I

    PCR Amplicon

    September 15, 200915

    Inactivate Enzymes with EDTA @ 65°C x 15 min

    (2 hr digest time)

  • Step 4: Bioanalyzer Lab-On-A-Chip Electrophoresis

    LOW

    MARKER

    HIGH

    MARKER A B C

    A

    B C

    September 15, 200916

    Prepare Fish ID Chip Add PCR-RFLP

    products

    Run on Agilent

    2100 Bioanalyzer

    Analyze

    Results

    • Thaw reagents • Prepare gel/dye mix

    • Prime chip

    • Select assay • Click START

  • Restriction Digest of Amplicons from Different Fish Samples

    P a

    c if

    ic C

    o d

    D o

    v e

    r

    T il

    a p

    ia (

    fa rm

    )

    S n

    a p

    p e

    r

    T ro

    u t

    T u

    n a

    P o

    s it

    iv e

    C o

    n tr

    o l

    S w

    a i

    C a tf

    is h

    T il

    a p

    ia (

    w il

    d )

    DdeI

    Hae III

    Nla

    III

    September 15, 200917

  • September 15, 200918

    Manual Entry or Automatic Import of Digest Products

    Step 5: RFLP Matcher Software

    Rank score output for each digest

    Combined score from all three digests identifies

    most-likely matching species

  • RFLP Patterns with Fish Admixtures

    Limit of detection in admixtures: 5%

    19 September 15, 2009

    Cod

    Haddock

    Mixture

  • Reproducibility Between Test Sites

    September 15, 200920

    Fish species /

    Test Site

    DdeI Hae III Nla III RFLP

    matcher

    score

    RFLP matcher species

    Trout

    Test Site A

    121, 353, 360

    121,354, 361

    38, 108, 327

    39, 109, 328

    100, 194

    100, 194

    0.867

    0.867

    Oncorhynchus clarki

    clarki

    Trout

    Test Site B

    119,351,357

    119,351,357

    _ 108,327

    _ 109,327

    99,192

    99,192

    1

    1

    Onc

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