Agilent Technologies Fish Species Identification System

Transcription

1 Agilent Technologies Fish Species Identification System AOAC Annual Meeting Exhibitor Presentation Scott Happe, Ph.D. Senior Research Scientist Stratagene Products Division 1

2 Preventing Fraud, Mislabeling, and Substitution: Primary Drivers for Fish Identification Testing Consumer Satisfaction/ Product Value Regulatory Compliance Sustainability and Environmental Protection Safety 2

3 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 Isoelectric focusing ELISA Objective results More resistant to sample processing Easy to perform High specificity/sensitivity PCR/QPCR RFLP Sequencing DNA-based methods 3

4 ELECTROPHORESIS Digest with Enzyme B Polymerase Chain Reaction (PCR) Restriction Fragment Length Polymorphism (PCR-RFLP) PCR: Amplify with generic fish primers PCR product: amplicon Sample DNA SPECIES SPECIES 2 1 4

5 Basis for the Fish Species Identification System is the PCR-RFLP Method Developed at Campden BRI Food Control 16 (7),

6 Agilent Bioanalyzer 2100 First commercially available Lab-on-a-Chip product (1999) Industry s ONLY microfluidicsbased 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) 6

7 Chip and Electrode Cartridge Molecular Assays On-chip gel electrophoresis for the analysis of RNA, DNA and proteins Disposable DNA chip Gel wells Ladder well Separation channel and point of detection 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 7

8 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 Agarose Gel Readout Bioanalyzer Readout 8

9 Agilent Fish Species ID Platform Extraction PCR RFLP Agilent 2100 Bioanalyzer < 1 hr ~ hrs 2.25 hrs <1 hr RFLP Matcher Software ~6 hrs total time from sample to results Fish Identification 9

10 Step 1: Extraction of DNA from Fish Tissue Using the Fish Species Identification System 1. Measure 40 mg 1 g tissue (fresh, frozen, or processed) Homogenization optional 2. Digest with Proteinase K Digestion Buffer Incubate at 65 o C 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 < 1 hr from tissue to DNA Page 10

11 DNA Yield, ug Total yield, µg Total yield, µg High DNA Yield from A Variety of Fish Samples Extractions from a Variety of Fresh Fish 16 Competitor Kits Agilent Kit Atlantic Salmon - wild Catfish - farm Pacific Cod - wild Tilapiawild Tilapiafarm Scallop - wild Trout Reinbow - farm Dover - wild Tuna - wild Salmon - farm Basa Swai - farm Snapper Pacificwild Yield range: 2-18 µg from 40 mg tissue Purity: Average of 2.19 A260/A280 PCR input requirement: ng Average 260/280: 1.41 Average 260/280: Yield from Alternative Fish Samples (40 mg each) 43µg µg 3.7µg Dry fish Canned fish Fish fins Page 11

12 PCR Target: 464 bp Region of the Fish cyt b Locus Polymorphic region flanked by conserved sequence 12

13 Step 2: PCR PCR Master Mix Fish Primer Mix PCR Cycling hrs cytb PCR Amplicon + 1 µl Fish DNA Extract OR Positive Control Salmon DNA 13

14 Pacific Cod Dover Swai Catfish Tilapia (farm) scallop Snapper Trout Tuna Tilapia (wild) Atlantic salmon Salmon (farm) Validation of Amplification from Different Fish Samples 14

15 Digest with Hae III Step 3: Restriction Digestion PCR Amplicon (2 hr digest time) Inactivate Enzymes with 65 C x 15 min 15

16 Step 4: Bioanalyzer Lab-On-A-Chip Electrophoresis Prepare Fish ID Chip Add PCR-RFLP products Run on Agilent 2100 Bioanalyzer Analyze Results Select assay Click START <30 min run Thaw reagents Prepare gel/dye mix Prime chip Load samples ( 1 µl) Vortex 1 minute LOW MARKER A B C HIGH MARKER C B A 16

17 Restriction Digest of Amplicons from Different Fish Samples Pacific Cod Dover Swai Catfish Tilapia (farm) Snapper Trout Tuna Tilapia (wild) Positive Control DdeI Hae III Nla III 17

18 Step 5: RFLP Matcher Software Manual Entry or Automatic Import of Digest Products Rank score output for each digest Combined score from all three digests identifies most-likely matching species 18

19 RFLP Patterns with Fish Admixtures Cod Haddock Mixture Limit of detection in admixtures: 5% 19

20 Reproducibility Between Test Sites Fish species / Test Site Trout Test Site A Trout Test Site B Trout Test Site C DdeI Hae III Nla III RFLP matcher score 121, 353, ,354, ,351, ,351, ,356, , 356, , 108, , 109, 328 _ 108,327 _ 109,327 39,109,331 39,110, , , ,192 99, , , RFLP matcher species Oncorhynchus clarki clarki Oncorhynchus clarki clarki Oncorhynchus clarki clarki Trout Test Site D 123,357, ,356,363 39,110,332 39,111, , , Oncorhynchus clarki clarki Trout QC specs 120, 350, , 108, , Oncorhynchus clarki clarki Mean 121, 354, , 109, , 194 SD 1.5, 2.4, , 1.0, , 1.4 (Cut-throat trout) 20

21 Agilent s Fish Species Identification System: Current Activities and Future Outlook Expanding authenticated profiles database (in conjunction with Steve Garrett s lab at Campden BRI): ~50 authenticated (gadoids, salmonids, flatfish) + 100s of theoretical Beta-testing of the Agilent Fish Species Identification System is underway with a major European seafood products manufacturer Quick adoption by staff new to DNA testing Expected commercial availability of the Agilent Fish Species Identification Kit is early 2010, shortly following the adoption of stricter import certification requirements in the EU 21

22 Acknowledgements Agilent Stratagene Products Division Harini Ravi Natalia Novorodovskaya Scott Basehore Jeff Braman Rachel Formosa Ronda Allen Rajesh Bagga Derek Hall Sarah Jandle Agilent Europe Nigel Skinner Juergen Schneider Stephen Mueller Martin Kratzmeier Campden BRI Steve Garrett Agilent Genomics Division Robert Kincaid Mary McBride For more information contact: Rachel Formosa Scott Happe 22

23 Thank you! 23