Overview on GMO detection approaches

Transcription

1 International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 1 Overview on GMO detection approaches Maddalena QUERCI Molecular Biology & Genomics Unit Institute for Health and Consumer Protection (IHCP) European Commission Joint Research Centre

2 Who uses detection methods and why? International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 2 GMO producers To assure purity and segregation of products To trace genetic modification in breeding Food & feed industry, seed companies To assure purity and segregation of products To assure compliance with legislation Competent (enforcement) authorities Product control, compliance with legislation To be able to retrieve specific products E.g. if marketing permission withdrawn Laboratories To provide analytical services to customers

3 Why to analyse GMOs? International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 3 Contained use Deliberate release into the environment Labelling and traceability Field experiments Premarket phase Marketing Postmarket phase

4 How to Know if a Product is Genetically Modified? International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 4 WT mrna mrna Protein enzyme Protein enzyme metabolites P E Gene T Construct metabolites GMO P E Gene T mrna mrna mrna Protein enzyme Protein enzyme Protein enzyme metabolites metabolites metabolites

5 How to Know if a Product is Genetically Modified? International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 5 Which method to select? Methods of analysis Biological whole plant Chemical fatty acids fibre structure Bioanalytical Protein DNA Phenotypical Genotypical

6 Detection strategies International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 6 Raw material processed target method product Soya DNA DNA Polymerase Polymerase Chain Chain Reaction Reaction (PCR) (PCR) Protein Protein Immuno Immuno assays assays Rape seed Fatty Fatty acid acid (HPLC/GC) (HPLC/GC)

7 Steps in DNA based GMO analysis International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 7 Sampling % GMO content M nt C Analytical DNA extraction Amplification (PCR)

8 Detection strategies, which method to select? International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 8 Experience along the production chain Increasing homogeneity / representativity Planted seed Harvest. crop Silo1 crop Silo2 crop Interm. Prod. 1 Interm. Prod. 2 Endproduct High sampling and subsampling effort Potentially decreasing analytical sensitivity due to processing

9 Operation of GMO laboratories to comply with EU legislation International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 9 GMO detection GMO? Yes/No Positive Negative GMO identification Yes No Illegal Are they Authorised? Yes/No GMO quantification Assay individual ingredients Must be Labelled? Yes/No < threshold No need for labelling > threshold Labelling required

10 Quantification of GMOs and labelling International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil % maize GM maize 0.50% 98.50% maize GM maize 1.50% 49.80% soybean maize GM soybean GM maize 0.20% 0.40% maize soybean 49.60% GM maize I GM maize II GM soybean No labelling Labelling No labelling required 98.80% 50% maize GM maize I GM maize II 0.80% 0.40% 48.80% soybean maize GM maize I GM maize II 0.80% 0.40% Ingredients How much maize is present? How much GMmaize 1 is present? Labelling required Labelling required How much GMmaize 2 is present? Make calculations

11 Detection strategies, which target to select? International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 11 Plant DNA P E Gene T Plant Plant DNA DNA HIGH 5. eventspecific 4. constructspecific eventspecific identification identification of gene construct SPECIFICITY 2. screening 3. genespecific identification of the gene GMO or not? LOW 1. speciesspecific Amplifiable DNA? Species?

12 GMO quantification by RTPCR International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 12 Wild type GMO Target taxon specific GM specific 1,60E00 1,60E00 1,40E00 1,20E00 NTC soya Soya stnd e Soya stnd f Soya stnd g Soya stnd h sample 1,40E00 1,20E00 NTC RRS stnd a RRS stnd b RRS stnd c RRS stnd d sample 1,00E00 1,00E00 8,00E01 8,00E01 6,00E01 6,00E01 4,00E01 4,00E01 2,00E01 2,00E01 0,00E ,00E ,00E01 PCR cycle no. 2,00E01 PCR cycle no. 1. Sample preparation, and DNA extraction 2. DNA amplification in realtime PCR machine GM endogenous Interpretation of result

13 Qualitative PCR: endpoint RTPCR detection International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 13

14 Pros and cons DNA methods International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 14 Genetic modifications = DNA modifications DNA stable and inheritable DNA traceable unit for all purposes Matrix limitations may apply Sensitive, fit for identification and quantification Costs: Efficient screening (multiple targets and GMOs) Expensive identification and quantification Equipment, reference material, skilled staff Limited coverage, although superior to protein Can only detect what we have methods for!

15 GMO analysis by immuno assays International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 15 Target Protein Antigen ELISA test Epitope Reverse ELISA Antibody LateralFlow Strip test

16 Pros and cons protein methods International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 16 Advantages/benefits: Speed Cost Practicability and easy transferibility Low risk of false positives (carry over) Well established in the food industry Drawbacks: Matrix limitations and sensitivity Coverage low (methods only for few GMOs) Low fitness for Qn analysis Limited identification (no event identification except for unique traits)

17 Present context International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 17 Worldwide adoption and use of GMOs is rapidly increasing (acreage, countries); Constant rise in GMO complexity, number of traits and events; In the EU: Mandatory labelling of GMOs and derived food/feed products (if above 0.9%) requires eventspecific methods; Postmarket monitoring requirements; GMO control based on combination of screening eventspecific detection methods; Increasing number of GMOs under approval; Asynchronous approval process complicates the analytical procedure. Higher number of methods to be applied for full product characterisation. Increased time and cost of analysis/sample.

18 Analysis of soybean samples International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 18 Homogenization of sample (soya) DNAExtraction repeat DNAExtraction if necessary Detection of target taxon sequence negative positiv Screening: 35SPromoter, further elements if necessary positive negativ GM specific detection ggf. GTS 4032 (RRsoya) Authorized within the EU No authorization within the EU, but positively safety assessed GU262 GU262 GU262 GU262? No authorization within the EU, no safety assessment done CaMV and / or Agrobacterium tumefaciens no transgenic DNA detected positive negative positive negative positive negative bezogen auf die untersuchten GVLinien Quantification Konzept zur Analytik von gentechnisch veränderten Futtermitteln, 2005, M. Egert et al.

19 International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 19 Next generation approaches for GMO analysis: Development and validation of highthroughput / multitarget detection methods Multiplex DNA microarray PCR1 PCR2 PCR3 PCR4 Genomic DNA Consensus primer PCR Slide with 2 arrays Food sample Extraction of DNA Selective amplification (marker sequences) and labelling B G Activated glass surface with fixed specific capture probe Hybridization of PCR products Analysis: presence of GMO specific sequences

20 International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 20 Next generation approaches for GMO analysis: Development and validation of highthroughput / multitarget detection methods Microsphere based assays (xmap/luminex technology)

21 International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 21 Within the present context. The only way to foster appropriate testing and to guarantee proper GMO control in the EU is to facilitate the work of enforcement laboratories. This can be achieved by developing and providing them tools able to overcome the difficulties of applying a complex analytical procedure, often exceeding laboratories capabilities.

22 International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 22 Next generation approaches for GMO analysis: Development and validation of highthroughput / multitarget detection methods Sample 1 RealTime PCR based readytouse multitarget analytical system for the detection of EU authorised and unauthorised GM events Sample 2 Targets: 7 plant species 39 GM events

23 International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 23 Realtime PCR based readytouse multitarget analytical system for GMO analysis Prespotted plates source: 48 realtime PCR methods validated by the CRL GMFF output: unique system in which all methods work with a unique set of conditions without loosing specificity and overall performance Doss ier Event Bt11 NK603 GA21 MON T H71 Sugar beet MON Cotton Cotton LLRICE62 Rice T45 oilseed rape EH Potato Ms8 Oilseed rape Rf3 Oilseed rape GT73 (RT63) Rapeseed LLCotton25 Cotton MON 531 Cotton A MIR604 Rf1 Rapeseed Rf2 Rapeseed Ms1 Rapeseed Topas 19/2 Rapeseed MON1445 Cotton Bt176 MON15985 Cotton 4032 GA21 MON88017 LY MON89788 MON89034 DP soybean MON88913 cotton Rice GM events P35S::bar LLRice601 Rice Bt63 Rice Bt10 Primer F Bt11 GCGGAACCCCTATTTGTTTA NK603 GA21 GTAGGATCGGAAAGCTTGGTAC TAGTCTTCGGCCAGAATGG MON863 ACAAGCGTGTCGTGCTCCAC GGGATAAGCAAGTAAAAGCGCTC 1507 TGGGATCTGGGTGGCTCTAACT T AGCTGGCGTAATAGCGAAGAGG MON810 CAATGGACACATGAATTATGC GTGTCAAAACACAATTTACAGCA MIR604 Bt176 CAGATTTTTGTGGGATTGGAATTC MON88017 TCCCATTCGAGTTTCTCACGT GCAAAAAAGCGGTTAGCTCCT LY038 GCGCACGCAATTCAACAG CTAAGGGAGGTCAAGATGTAGC 3272 Species Sugar beet Cotton Cotton Rice Rape Potato Rape Rape Rape Cotton Cotton Soy Rape Rape GGGTGAGACAATATATCGACG Rape ACGCTGCGGACATCTACATT MON89034 Rape Cotton Cotton Soy TCATCAGACCAGATTCTCTTTTATGG MON89788 TCCCGCTCTAGCGCTTCAAT Soy Cotton Rice Rice Rice ATGAATGACCTCGAGTAAGCTTGTTAA CTTATCGTTATGCTATTTGCAACTTTAGA TCGAAGGACGAAGGACTCTAACGT CTCATTGCTGATCCATGTAGATTTC AAATATTAACAATGCATTGAGTATGATG GTTAGAAAAAGTAAACAATTAATATAGCCGG AGCATTTAGCATGTACCATCAGACA CCATATTGACCATCATACTCATTGCT GTTGCGGTTCTGTCAGTTCC Bt10 GGCCGTGAACGAGCTGTT A GGAGTAAGACGATTCAGATCAAACAC GTTACTAGATCGGGGATATCC TTCATTCAAAATAAGATCATACATACAGGTT CGTTATGCTATTTGCAACTTTAGAACA GAGCAGGACCTGCAGAAGCT TGGGTTCAGTCTGCGAATGTT TTCTCCATATTGACCATCATACTCATT DP GTCGAATAGGCTAGGTTTACGAAAAA GGCTTTGGCTACCTTAAGAGAGTC TATCCTTCGCAAGACCCTTCC Potato TCTAGGATCCGAAGCAGATCGT GACTGCTGGAGTGATTATCGACAGA EH CACACAGGAGATTATTATAGGGTTACTCA Primer R Oilseed rape Probe T45 Ms8 Rf3 GT73 Rf1 Rf2 Ms1 Topas 19/2 Rice LLRICE62 LLRice601 Bt63 Rice Sugar beet H71 Sugar beet Cotton MON1445 CCTTTTCCATTTGGG MON88913 LLCotton25 MON MON15985 TCCAAGAATCCCTCCATGAG AAGAGATAACAGGATCCACTCAAACACT TGGCTCGCGATCCTCCT TGTTACGGCCTAAATGCTGAACT CTTTGCCAAGATCAAGCG GACATGATACTCCTTCCACCG CCTTAATTCTCCGCTCATGATCAG AATGCTGCTAAATCCTGAG GCCACCTTCCTTTTCCACTATCTT GGACAATGCTGGGCTTTGTG ACTCTTTCTTTTTCTCCATATTGACC TGCTAACGGGTGCATCGTCTA GACTCTGTATGAACTGTTCGC TCCCTTAATTCTCCGCTCATGA GGAGGGTGTTTTTGGTTATC CATAAAGGAAGATGGAGACTTGAG GCTTATACGAAGGCAAGAAAAGGA CAAGGAACTATTCAACTGAG AACCAATGCCACCCCACTGA ATTCAGGCTGCGCAACTGTT GGTCATAACGTGACTCCCTTAATTCT CGGGCCTAACTTTTGGTGTG GGGCATCGCACCGGTGAG CTAGATCGGAAGCTGAAGATGG CGACCGGCGCTGATATATGA ATCGACCTGCAGCCCAAGCT GGGAAGAAGCCTACATGTTTTCTAA AAGGTTGCTAAATGGATGGGA GGCATTTGTAGGAGCCACCTT GCGATCCTCCTCGCGTT TCCGGAGTTGACCATCCA AGGAATTCGATATCAAGCTTATCGA CGTTTCCCGCCTTCAGTTTA TCGAGCAGGACCTGCAGAA CGGTATCTATAATACCGTGGTTTTTAAA TTTGATATTCTTGGAGTAGACGAGAGTGT CAAATTACCCATTAAGTAGCCAAATTAC ATGTCGGCCGGGCGTCGTTCTG GGAGGGCGCGGAGTGT AGCTCGGTACCTCGACTTATTCAG ACACGGAAATGTTGAATACTCATACTCT AAATACATTCAAATATGTATCCGCTCA TGGTACCACGCGACACACTTCCACTC CATATACTAACTCATATCTCTTTCTCAACAGCAGGTGGGT TGAACACCCATCCGAACAAGTAGGGTCA TAACTCAAGGCCCTCACTCCG TCATTGAGTCGTTCCGCCATTGTCG TTTAAACTGAAGGCGGGAAACGACAA AAGGCGGGAAACGACAATCT AACATCCTTTGCCATTGCCCAGC TTGGGTTAATAAAGTCAGATTAGAGGGAGACAA TACTCATTGCTGATCCATGTAGATTTCCCG CGCACCGATTATTTATACTTTTAGTCCACCT TAGAGGACCTAACAGAACTCGCCGT AGATTGTCGTTTCCCGCCTTCAGTT AATATAATCGACGGATCCCCGGGAATTC CGCACGCTTATCGACCATAAGCCCA TTCCCGGACATGAAGATCATCCTCCTT CTTAACAGTACTCGGCCGTCGACCGC TTGTCCCTCCACTTCTTCTC CGGTCCTCCGATCGCCCTTCC AGGCGGGAAACGACAATCTGATCATG CTCATCATCCTCACCCAGTCAGCATCA CACCGGCCAAATTCGCTCTTAGCCGT CTCATTGCTGATCCACCTAGCCGACTT TCCCGCGTCATCGGCGG ATCAGATTGTCGTTTCCCGCCTTCAGTTT AGCAACCAGATCGGCCGACACC CCGCTCTAGAACTAGTGGATCTGCACTGAA TTTCTCAACAGCAGGTGGGTCCGGGT TCCCGCCTTCAGTTTAAACAGAGTCGGGT CGAGCGGAGTTTATGGGTCGACGG ACTGCTGACGCGGCCAAACACTG CTGAAGGCGGGAAACGACAATCTG ATCCCCGGAAATTATGTT CTCTAGAGATCCGTCAACATGGTGGAGCAC AACTATCAGTGTTTGACTACAT TCTATATAAGGAAGTTCATTTCATT CCACCTCCCAACAATAAAAGCGCCTG TCGAGTTCATTCCAGTTACTGCAACACTCGAG AATAACCCTGATAAATGCTTCA Probe MGB MGB MGB MGB MGB Report er

24 International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 24 Detection of cotton event MON15985 E2 = SAH7 cotton reference gene method G2 = MON531 eventspecific method G11 = MON15985 eventspecific method

25 International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 25 Detection of maize event Bt10 A1 = maize reference gene method D12 = Bt10 eventspecific method

26 Performance of eventspecific plates: specificity International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 26 SAMPLES WILD TYPE GM EVENTS Cotton Rice Rapeseed Sugar beet Potato Bt11 NK603 GA21 Monsanto MON T H71 Sugar beet MON X Cotton LLRICE62 Rice T45 Rapeseed EH Potato Ms8 Rapeseed Rf3 Rapeseed GT73 (RT63) Rapeseed LLCotton25 Cotton MON 531 Cotton A MIR604 Rf1 Rapeseed Rf2 Rapeseed Ms1 Rapeseed Topas 19/2 Rapeseed MON 1445 Cotton Bt176 MON Cotton 4032 GA21 Syngenta T ype of detec tion M ethods MON LY Taxon specific E ven t sp e cific HMG R SAH7 Cotton R PLD Rice R CruA Rapeseed R Lectin R GS Sugar beet R UG Pa se P otato R Bt11 NK603 M aize GA21 M aize Method 1 MON M aize T25 M aize M aize H71 Sugar beet MON Cotton Cotton LLRICE62 Rice T4 5 Rape seed EH Potato M s8 R apese ed Rf3 Rapeseed G T73 R apese ed LLCOTTON25 Cotton M O N 53 1 Cotton A S oybe an MIR604 Rf1 Rapeseed Rf2 Rapeseed M s1 R apese ed Topas 19/2 Rapeseed M ON 1445 Cotton Bt176 M ON Cotton GA21 M aize Method 2 MON LY 038 M aize 3272 M aize MO N S oybean MON DP S oybe an M ON Cotton Construct specific Rice P35S::bar E ven t sp e cific LLRice601 Rice Bt63 Rice Bt10

27 Performance of eventspecific plates: sensitivity International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 27 Input DNA = 100 ng/well GM content = 0.045% each event Threshold setting = optimised Confidence = 95% (10 repetitions) HM G Cotton Rice OSR Soya SB P otato t11 NK603 GA21 M ON T H71 MON LL62 T45 EH92 Ms8 Rf3 GT73 LL A2704 M IR604 Rf1 Rf2 Ms1 Topas 1445 Bt GA21 syn LY DP P35:BAR LL601 Bt63 Bt10 Ct values

28 International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 28 Workflow and approximate timing for GMO analysis using the readytouse multitarget analytical system 1 Extract DNA 2 Add TaqMan Universal PCR Master Mix and load the plate 3 Perform RealTime PCR amplification 4 Analyse data 1 4 hrs 15 min 2 2½ hrs

29 International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 29 Readytouse prespotted plate/strip systems in response to the different needs of GMO analysis: Cropspecific formulation (for commodities testing) and soybean events detected Plate layout well RTiPCR method well RTiPCR method A1 A2 HMG Ref HMG Ref B1 B2 LY A B HMG Ref LY038 HMG Ref 3272 Bt11 MON89034 NK GA21 Lectin Ref MON863 Lectin Ref DAS1507 A T DAS59122 MON89788 MON810 DP MIR604 DP MON88017 A A3 Bt11 B3 MON89034 C HMG Ref HMG Ref Bt11 NK603 GA21 MON863 DAS1507 T25 DAS59122 MON810 MIR604 MON88017 A4 A5 A6 A7 A8 A9 NK603 GA21 MON863 DAS1507 T25 DAS59122 B4 B5 B6 B7 B8 B Lectin Ref Lectin Ref A MON89788 D E F G H LY038 HMG Ref LY038 HMG Ref LY HMG Ref 3272 HMG Ref 3272 MON89034 Bt11 MON89034 Bt11 MON NK NK Lectin Ref GA21 Lectin Ref GA21 Lectin Ref Lectin Ref MON863 Lectin Ref MON863 Lectin Ref A DAS1507 A DAS1507 A T T MON89788 DAS59122 MON89788 DAS59122 MON89788 DP MON810 DP MON810 DP DP MIR604 DP MIR604 DP A MON88017 A MON88017 A A10 MON810 B10 DP A11 MIR604 B11 DP A12 MON88017 B12 A

30 International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 30 Readytouse prespotted plate/strip systems in response to the different needs of GMO analysis: Screening formulation based on matrix approach Requirements to apply screening in GMO analysis: defined analyte type (DNA) defined GMO Universe (e.g. EU authorized GMO for food and feed use) defined targets in the GMO of this Universe validated methods to demonstrate the presence of these targets

31 Matrix approach in GMO screening International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 31 Relationship between the GMO Universe, the targets and the methods Validation GMO Universe Method 1 Method 2 Method 3... Targets 1 Targets 2 Targets 3... Screening GMO 1 GMO 2 GMO 3 GMO 4 GMO 5 GMO 6 GMO 7...

32 International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 32 Readytouse prespotted plate/strip systems in response to the different needs of GMO analysis: Screening formulation based on matrix approach What is a GMO Matrix (for screening purposes) 1) A description of the set of GMO in form of a (Excel) table 2) Each GMO is described as a combination of genetic elements which are the targets for the screening (such as the p35s, tnos, CryIAb,...) 3) A (mathematical) matrix form wherein the relation between targets and GMO is represented the (molecular) targets are listed as columns the GMO are represented as rows

33 International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 33 Screening formulation based on prespotted plates and Matrix approach The matrix representation facilitates to describe the relationships between the GMO Universe, the targets and the methods within a screening set up Method 1 Method 2 Method 3 Target 1 Target 2 Target 3 Target 4 Target 5 Target 6 GMO1 GMO2 GMO3 GMO4 GMO5 ': present ': absent

34 International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 34 COSYPS: Combinatory SYBR Green QPCR Screening Semiquantitative detection system using a set of SYBR Green RTPCR methods coupled to a software based application for data interpretation X Prime PCR Test Core element class Primer Reference 3 RBCl Plant Debode (pers. Comm.), Lectin Species (soya) Terry and Harris, Alcohol dehydrogenase Species (maize) SBB/ISP 11 Cruciferine Species (Oilseed rape) SBB/ISP 13 CaMV p35s Generic (promotor) SBB/ISP 17 Agrobacterium TNOS Generic (terminator) SBB/ISP 19 CP4EPSPS Trait (herbicide res.) SBB/ISP 23 CryIAb Trait (insect res.) SBB/ISP 29 PAT/pat Trait (herbicide res.) SBB/ISP 31 PAT/bar Trait (herbicide res.) SBB/ISP GMO p35s tnos CP4 EPSPS PAT/pat PAT/bar Cry1Ab GTS 40/3/2 X X X Bt 11 X X X X Bt 176 X X X MON 810 X X X GA 21 X T25 X X NK 603 X X X MON 863 X X TC1507 X X DAS59122 X X Bt10 X X X X GT73 X MS1/RF2/ MS1xRF2 X X MS1/RF1/ MS1xRF1 X X MS8/RF3/ MS8xRF3 X X TOPAS 19/2 X X T45 X X Falcon GS 40/90 X X MON 1445 X X X MON 531 X X X LLRICE601 X X Bt63 X X RUR H71 X

35 International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 35 Combination of screening methods targeting common GM elements GM 1 GM Sample RESULTS (1 method = 1 element) METHOD n. GM Interpretation GM GM Sample 1 GM 1 GM 5 GM Sample 2 Sample 3 Sample 4 GM 2 GM 3 GM 4 GM Sample 5 GM 5 Sample 6 GM 3 GM 5 Sample 7 GM 6 Sample 8 GM 5 GM 6 Sample 9 NO GM Sample 10 GM 1 GM 5 GM 6 Sample 11 GM 1 GM 6?

36 Well Sample N a Detector Name Reporter Ct 1 HMG Cotton Undetermined 3 Rice Undetermined 4 OSR Undetermined 5 Soya SB Undetermined 7 Potato Undetermined 8 Bt11 Undetermined 9 NK GA21 Undetermined 11 MON863 Undetermined Undetermined 13 T25 Undetermined Undetermined 15 H71 Undetermined 16 MON Undetermined Undetermined 19 LL62 Undetermined 20 T45 Undetermined 21 EH92 Undetermined 22 Ms8 Undetermined 23 Rf3 Undetermined 24 GT73 Undetermined 25 LL25 Undetermined Undetermined 27 A2704 Undetermined 28 MIR 604 Undetermined 29 Rf1 Undetermined 30 Rf2 Undetermined 31 Ms1 Undetermined 32 Topas Undetermined Undetermined 34 Bt176 Undetermined Undetermined GA21 Undetermined Undetermined 39 LY038 Undetermined Undetermined Undetermined Undetermined 43 DP3560 Undetermined Undetermined 45 P35:BAR Undetermined 46 LL601 Undetermined 47 Bt63 Undetermined 48 Bt10 Undetermined International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 36 Decision Support Systems in GMO analysis using a matrixbased approach in combination with readytouse multitarget analytical system. 1. Field or sample to be tested 2. DSS Level 1 Matrix approach GM GM GM GM GM 5 9 GM 6 5 GM Sample Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11 METHODS GM INTERPRETATION 1) Sample definition, 2) Establishment of a GMO matrix and decision on optimal analysis strategy (screening & identification), 3. DSS Level 2 Application of analytical method(s) 3) RTiPCR amplification using readytouse prespotted plates, and 4) Combined interpretation of the analytical results. 4. DSS Level 3 Data analysis & interpretation Sample Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample RTPCR RESULTS GM INTERPRETATION GM1 GM2 GM3 GM4 GM5 GM3 GM5 GM6 GM5 GM6 NO GM GM1 GM5 GM6 GM1 GM6?

37 International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 37 Future technological impact The readytouse multitarget analytical system based on prespotted plates has demonstrated a great potential for increasing harmonisation in GMO testing: Tool to test all (several) many events at once (need to constant updating) Unique tool/provider for all control laboratories; Harmonised set of targets / methods; Flexibility to be adapted according to needs; Same tool if used by different laboratories comparable results. The combination of this system with the matrixbased screening approach, integrated into a Decision Support System allows to tackle the current complexity and will foster harmonisation GMO analysis

38 International Workshop on Harmonisation of GMO detection and Analysis for Central and South America 45 December 2009 Foz do Iguazu, Brazil 38 Thank you! Gracias por su atenciòn! Muito obrigada!