agilent technologies fish species identification system agilent technologies fish species...

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

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fa rm

)

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A tl

a n

ti c

s a

lm o

n

S w

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C a

tf is

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S a

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fa rm

)

T il

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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

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T il

a p

ia (

fa rm

)

S n

a p

p e

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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