Upload
phamlien
View
214
Download
0
Embed Size (px)
Citation preview
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
2
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
October 26, 2011Public
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
October 26, 2011Public
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”
October 26, 2011Public
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
October 26, 2011Public
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:
October 26, 2011Public
8
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
October 26, 2011Public
9
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)
October 26, 2011Public
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.
October 26, 201111Public
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
October 26, 2011Public
12
< 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
October 26, 2011Public
13
< 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
October 26, 2011Public
14
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
October 26, 2011Public
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
October 26, 201116Public
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
Page 18 October 26, 2011Public
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
Page 19
0
Dry fish Canned fish F ish fins
October 26, 2011Public
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
20 October 26, 2011Public
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
21 October 26, 2011Public
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
October 26, 2011Public
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
24
• Prime chip
October 26, 2011Public
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 )
25
(electronic records)
October 26, 2011Public
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
26 October 26, 2011Public
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
27 October 26, 2011Public
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
28 October 26, 2011Public
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
29
Combined score from all three digests identifiesmost-likely matching species
October 26, 2011Public
Pattern Matching: RFLP Report
Page 30 October 26, 2011Public
RFLP Patterns with Fish Admixtures
CodCodHaddockMixture
Limit of detection in admixtures: 5%Limit of detection in admixtures: 5%
31 October 26, 2011Public
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
32 October 26, 2011Public
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.
October 26, 2011Public
33
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
October 26, 201134Public
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 )
October 26, 201135Public
Thank you!Thank you!
WATCH THE VIDEO: https://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1291
October 26, 201136Public