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Agilent TechnologiesAgilent Technologies DNA Fish ID Solution

Agilent TechnologiesLenore Kelly Ph DLenore Kelly, Ph.D.

1

Agilent Technologies – Introduction

Spun off Hewlett-Packard in 19993 major fields of business: Electronic Measurement, Life Sciences and Chemical AnalysisHeadquarters in Santa Clara (US)EU headquarters Waldbronn (Germany) with officesin many EU countries

October 26, 2011Public

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Agilent Technologies – A Focus on MeasurementT ti th h lfTesting more than half of the world’s 4 billion

cell phonesEquipping 200+ communications

Keeping our air, water soil and food

Ad i

communicationsservice providers

water, soil and foodclean and safe

Advancingnext-generation

integratedvoice, video & data

Aiding the discoveryand quality

of medicinesof medicines

Analyzing thecauses and cures

Making the worldmore safe and secure

Enabling the militaryto be more flexible,

bil d li bl

causes and curesfor diseasefrom crime and drugs

Agilent Profile

mobile and reliable

Authenticity – Important Factor in the Market

What do I have in my fish product?Multiple methods available todayProtein or DNA basedApplication in analysis of fraud, mislabelingpp y , gor substitution

Where does the fish come from?Scientifically more challenging – Notreadily availableTrace minerals fatty acid analysis (MS)Trace minerals, fatty acid analysis (MS)or DNA based using polymorphismsNo general approach available – solutionson a species by species basis


4

Seafood Identification – Increased Importance in the Market

Protein or DNA based MethodsProtein or DNA based MethodsUsed in analysis of potential fraud,mislabeling or substitution cases

Drivers for testing:

L i l ti S t i bilitLegislation

Industry

C

SustainabilitySupply Chain Management

Product value,Consumers Product value,Consumer Satisfaction


5

Mislabeling and Fraud – Large Economic ImpactMislabeling and substitution have large economic consequences:Mislabeling and substitution have large economic consequences:

Fully labeled products (species/catch area) allow informed consumer choice andare a marketing tool

Premium species get substituted by low cost/lower quality species

Protecting brands and avoiding liabilities resulting from fraud in thesupply chain needs a reliable robust method of monitoring productssupply chain needs a reliable, robust method of monitoring productsand suppliers

2010 Univ College Dublin Ireland

2010 NJ.com , USA

2007 Chicago Sun-Times , USA

DNA tests done on sushi described as red snapper

2010 CBC news , Canada

„DNA testing of a variety of fish bought at severalCanadian grocery stores…some species being soldare endangered…22% were mislabeled

2010 Univ. College Dublin, Ireland

„25% of fish products labeled and sold as cod

„Sterling Seafood founder admits $60M tariff fraud bymislabeling catfish imports”


6

„DNA tests done on sushi described as red snapper…from 14 restaurants…not one was really red snapper“ and haddock [in Ireland] are from different species”

Fish Identification Methods – Morphologyf f• Identification based on shape, color, scales, fins

• Requires expert for identification• Only applicable to whole, non-processed fish

Morphology

Pictures from www.fishbase.org


7

Fish Identification Methods – Protein based• Analysis of protein patterns

I l t i f i (IEF) i AOAC ffi i l th d 980 16• Isoelectric focusing (IEF) is AOAC official method 980.16

• Inexpensive and simple to perform• Proteins are highly susceptible to heat treatment and other processing steps• Limited differences of profiles between speciested d e e ces o p o es be ee spec es• Difficult to interpret and highly subjective, experienced user needed• Usually requires running an authenticated sample in parallel• Does not work with mixtures

Protein-based methods

Regulatory Fish Encyclopedia:


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Regulatory Fish Encyclopedia:U.S. Food and Drug Administration, 1993-2009

Fish Identification Methods – DNA-based• Objective resultsObjective results• More resistant to sample processing• Easy to perform• High specificity/sensitivity

DNA-based methods

SequencingHigh initial investment (> 100K $)High maintenance costsgNot usable with mixed samplesMethod of choice for new species

PCR-RFLPPCR RFLPDiscrimination of highlyrelated speciesWorks with mixturesLow initial setup costs


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Low initial setup costs

Fish Identification Methods – PCR RFLPMitochondrial target: High copy numberg g pyCommonly used Cytochrome b (Cytb)or Cytochrome Oxidase I (COX1) gene (Barcode of Life)Cytb sequence information well covered for fish species

Extract DNAAmplify region ofinterest on DNA

Fragment DNA to generatespecific patterns

PCR RFLP Workflow

Food Control 16 (7), 601-607, (2004)

PCR RFLP Method is an accredited/approvedmethod in many countries

http://www chem agilent com/Library/

J Agric Food Chem 53 (9), 3388-3357 (2005)

method in many countriesTransfer of method to 2100 Bioanalyzer forimproved analysis published 2004 and 2005by Stephen Garrett from Campden BRI (UK)


10

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

Agilent’s DNA Fish ID Solution

The Agilent DNA Fish ID solution is a simple, fast, and accurate DNA- based screening method to identify g yfish species in fresh and processed seafood samples.

Enhance the quality of seafood products.

Reduce the environmental impact of illegal fishing activities.

Maintain accurate records suitable for compliance with governmental regulations.


Agilent DNA Fish ID Solution - WorkflowSample Amplification of Generation of speciesSample

purificationAmplification ofspecies ID target

Generation of speciesspecific patterns

< 1 h ~ 1 5 h ~2 5 h< 1 h(depends on # samples)

~ 1.5 h ~2.5 h

Pattern analysisusing RFLP Decoderusing RFLP Decoder

< 1 h6 h from sample to result


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< 1 h(depending on # samples)

~ 6 h from sample to result

The 2100 Bioanalyzer Capillary Electrophoresis systemsystem

2100 BioanalyzerLab-on-a-chip system

Traditional slab gelelectrophoresis

Preparation of chip (5 - 15 min):Simple easy to follow procedureLow sample volume (1 µl)

Prepare and run gel (1 - 3 h):Larger sample volume needed

Run and Detection (30 min):Highly sensitive (0 1 ng of DNA)

Stain, destain gel and imageacquisition (0.5 – 1 h):Highly sensitive (0.1 ng of DNA)

Data in digital formatacquisition (0.5 1 h):

Low sensitivity (>50 ng DNA) Image needs to be digitalized

Gel analysis

Analysis:Automated analysis (sizing and quantitation)Comparison between samples and runsHigh resolution (5 bp difference)+ accuracy (± 10%)

Analysis:Resolution restricted High sizing variability

No accurate quantitative information

1 h > 2 h


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< 1 h > 2 h

Interpretation of Results – RFLP Decoder Software

Pattern 1(Dde I)

Pattern 2(Hae III)

Pattern 3(Nla III)(Dde I)

scoring panel(Hae III)

scoring panel(Nla III)

scoring panel

Sample selectionCombined score: Histogram:Indicates most likely species

Score value indicates quality of matchSeparation of top scoring speciesand other hits creates confidencein result


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Influence of processing on DNA quality

Fresh fish (trout):Avg. size 10K bp

The kit works well with a wide variety of fish samples:

Fried fish (whitefish):

Fresh or frozen fish

Cooked (or fried) fishFried fish (whitefish):Avg. size 11k bp

Smoked fish

Salted fishCanned fish (sardines):Avg. Size 122 bp!!!

Salted fish

Dried fish

C d fi h i diffi lt d t th hi h l l f DNACanned fish is difficult due to the high level of DNAfragmentation caused by the various processing steps


15

Agilent DNA Fish ID Solution:Current Activities and Future Outlook

• Validating experimentally-derived profiles database (in conjunction with Steve Garrett’s lab at Campden BRI):

C tl 40 th ti t d ( d id l id fl tfi h) 100 f th ti lCurrently ~40 authenticated (gadoids, salmonids, flatfish) + 100s of theoretical

• Utilization of the Agilent Fish Species Identification System is underway with several groups including a major European seafoodunderway with several groups, including a major European seafood products manufacturer

Quick adoption by staff new to DNA-based testing

• The Agilent Fish Species Identification Kit is shipping


how exactly does it work?

17 October 26, 2011Public

Step 1: Extraction of DNA from Fish Tissue Using the Agilent DNA Fish ID Solution

1. Measure 40 mg – 1 g tissue (fresh, frozen, or processed)Homogenization optional

g

Homogenization optional

2. Digest with Proteinase K Digestion BufferIncubate at 65oC for 10 minIncubation Time: 10 minTotal Time: 15 min

3. Capture DNA onto glass-fiber spin columnp g p5 min

4. Wash with High Salt Buffer (1X)Wash with 80% Ethanol (3X)Wash with 80% Ethanol (3X)5 min

5. Elute DNA

< 1 hr fromtissue to DNA

5 min


High DNA Yield from A Variety of Fish Samples

14

16

18

d, µ

g

g

12

14

16Competitor Kits Agilent KitExtractions from a Variety of Fresh Fish

4

6

8

10

12

Tota

l yie

l

Tota

l yie

ld, µ

g

4

6

8

10

0

2

AtlanticSalmon -

wild

Tilapia-wild

Catfish -farm

PacificCod -wild

Tilapia-farm

Scallop -wild

TroutReinbow

- farm

Dover -wild

Tuna -wild

Salmon -farm

BasaSwai -farm

SnapperPacific-

wild

Yield range: 2 18 µg from 40 mg tissue

0

2

Average 260/280: 1.41

Average 260/280: 2.09

Yi ld f Alt ti Fi h S lYield range: 2-18 µg from 40 mg tissuePurity: Average of 2.19 A260/A280 PCR input requirement: 0.5 – 500 ng

25

30

35

40

Yield from Alternative Fish Samples(40 mg each)

d, u

g43µg

0

5

10

15

20

DN

A Yi

eld

6.6µg 3.7µg

0

Dry fish Canned fish F ish fins


Step 2: PCR

PCR Master Mix

cytb

PCR Amplicon

PCR Cycling

1.5-2 hrs

Fish Primer Mix

+ 1 µl Fish DNA ExtractOR

Positive Control Salmon DNA


Validation of Amplification from Different Fish Samples

fic C

od

er ia (f

arm

)

op pper

t ic s

alm

on

sh on (f

arm

)

ia (w

ild)

Paci

f

Dov

e

Tila

pi

scal

lo

Snap

Trou

t

Tuna

Atla

nt

Swai

Cat

fis

Salm

Tila

p i


Step 3: Restriction Digestion

PCR Amplicon

with

III

Dig

est w

Hae

I

Inactivate Enzymes with EDTA @ 65°C x 15 min

(2 hr digest time)

22

Inactivate Enzymes with EDTA @ 65 C x 15 min


Polymerase Chain Reaction (PCR) –Restriction Fragment Length Polymorphism (PCR-RFLP)

PCR: Amplify with generic

fi h iPCR product:“ li ”

st w

ith

yme

B

fish primers

Sample DNA

“amplicon”

Dig

esEn

zy

S

SPECIES 1SPECIES 2

PHO

RES

ISLE

CTR

OP

EL23 October 26, 2011

Public

Step 4: Bioanalyzer Lab-On-A-Chip Electrophoresis Prepare Fish ID Chip Add PCR RFLP Run on Agilent AnalyzePrepare Fish ID Chip Add PCR-RFLP

productsRun on Agilent

2100 BioanalyzerAnalyzeResults

• Select assay• Click START• <30 min run

ABC

C

A

B C

• Thaw reagents• Prepare gel/dye mix

P i hi

•Load samples ( 1 µL)•Vortex 1 minute

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• Prime chip


Agilent Bioanalyzer 2100 Fi t i ll il bl• First commercially available Lab-on-a-Chip product (1999)

• Industry‘s ONLY microfluidics• 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 ( l i d )

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(electronic records)


Chip and Electrode Cartridge Molecular AssaysOn-chip gel electrophoresis for the analysis of RNA, DNA and proteins

Gel wells

Disposable DNA chip

Ladder wellSeparation channel and point of detection

Chi d t 12 l ll l ll• Chips accommodate 12 sample wells, gel wellsand a well for a sizing standard (ladder)

• Gel matrix is injected into etched channelson the chipS l i d ith i k d l d d• 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 ti h l d t th d t tseparation channel and past the detector


Bioanalyzer Instrumentation Enhances Ease-of-Use and Accuracy of Pattern MatchingEase 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

Bi l R d tg

Agarose Gel Readout

Bioanalyzer Readout


Restriction Digest of Amplicons from Different Fish Samples

d rm)

ontr

ol

ld)

Paci

fic C

od

Dov

er

Tila

pia

(far

Snap

per

Trou

t

Tuna

Posi

tive

Co

Swai

Cat

fish

Tila

pia

(wil

DdeI

Hae III

Nla III


Manual Entry or Automatic Import of Digest ProductsStep 5: Pattern Matching with RFLP Decoder Software

Rank score output for each digest

Combined score from all three digests identifies

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Combined score from all three digests identifiesmost-likely matching species


Pattern Matching: RFLP Report


RFLP Patterns with Fish Admixtures

CodCodHaddockMixture

Limit of detection in admixtures: 5%Limit of detection in admixtures: 5%


Reproducibility Between Test SitesFish species /

Test SiteDdeI Hae III Nla III RFLP

matcher score

RFLP matcher species

Trout 121, 353, 360 38, 108, 327 100, 194 0.867 Oncorhynchus clarki

Test Site A 121,354, 361 39, 109, 328 100, 194 0.867 clarki

Trout

Test Site B

119,351,357

119,351,357

_ 108,327

_ 109,327

99,192

99,192

1

1

Oncorhynchus clarki clarki

Trout

Test Site C

122,356,363

122, 356, 363

39,109,331

39,110,330

100,195

101,196

1

1

Oncorhynchus clarki clarki

Trout 123,357,364 39,110,332 100,194 1 Oncorhynchus clarki

Test Site D 122,356,363 39,111,331 101,195 1 clarki

Trout QC specs 120, 350, 358 40, 108, 327 101, 194 0.867 Oncorhynchus clarki clarki

(C t th t t t)(Cut-throat trout)Mean 121, 354, 361 39, 109, 329 100, 194

SD 1.5, 2.4, 2.8 0.4, 1.0, 2.1 0.8, 1.4


Summary of Agilent DNA Fish ID Solution

Ease of use and simplicityConveniently packaged reagents combined with Bioanalyzer readout andRFLP pattern matching software.Database of species specific patterns removes need for authenticatedp p psample in the same run

Fast results from a broad range of samplesFast results from a broad range of samplesSample to result in just one working day thru streamlined and simplifiedprotocols.Works with raw, cooked, smoked, dried and salted fish

Accurate and reliable resultsBased on a trusted method approved and accredited in many countriespp yUser-expandable database of experimental and theoretical profiles for > 200fish species.


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Agilent DNA Fish ID Solution:Ordering Information

• Agilent DNA Fish ID Ensemble - 5500-0100• Agilent DNA Isolation Kit - 5500-0051

Agilent PCR RFLP Fish ID Kit 5500 0001

g

• Agilent PCR-RFLP Fish ID Kit - 5500-0001• Agilent DNA 1000 Kit - 5067-1504

• Agilent RFLP Decoder Software, Fish - G5301A

• Agilent 2100 Bioanalyzer with chip priming station and IKA vortex mixer –G2939AA


Acknowledgements

Agilent Stratagene Products DivisionHarini RaviNatalia Novorodovskaya

Agilent EuropeNigel SkinnerJuergen SchneiderNatalia Novorodovskaya

Scott BasehoreJeff BramanRachel Formosa

Juergen SchneiderStephen MuellerMartin KratzmeierKnut Wintergerst

Ronda AllenRajesh BaggaDerek Hall

g

Campden BRISarah Jandle

Agilent Labs DivisionRobert Kincaid

Steve Garrett

For more information contact:Rachel Formosa ([email protected])Robert Kincaid

Mary McBride

( @ g )


Thank you!Thank you!

WATCH THE VIDEO: https://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1291


agilent fish id webinar.pptx [read-only] fish id... · clean and safe advancing next-generation...

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