A practical approach to screen for authorised and non-authorised GMOs

Transcription

1 A practical approach to screen for authorised and non-authorised GMOs Dr. Lutz Grohmann Federal Office of Consumer Protection and Food Safety Berlin, Germany 2 nd Workshop on Harmonisation of GMO analysis September 2010, Zagreb, Croatia

2 Federal Office of Consumer Protection and Food Safety Management Authority for Health-Related Consumer Protection 5 Departments, 470 Employees Food, Feed and Commodities Plant Protection Products Veterinary Drugs Genetic Engineering Analysis Laboratories (NRLs, EURL for Pharmacologically Active Substances) Headquarters in Braunschweig and Berlin

3 Federal Office of Consumer Protection and Food Safety National Competent Authority in the GMO Notification Process Directive 2001/18/EC (Authorisation of Deliberate Release) Regulation (EC) 1829/2003 (Placing on the Market, Cultivation of GMO) Publication of an Official Collection of Analytical Methods Art. 64 Food & Feed Law (with experts from enforcement, industry & research) develop & ring trial validation of GMO detection methods contribute to harmonisation and standardisation ENGL / DIN, CEN, ISO National Reference Laboratory for GM food & feed according to Regulation (EC) No. 882/2004 member of European Network of GMO Laboratories (ENGL)

4 Detection and control The Federal German Länder are responsible for inspection and control of the contained use of GMO, of field trials and of placing on the market of GMOs as well as the products derived from GMO For official monitoring of food and feed the Federal Länder take samples at food companies or from the market place Sampling and detection methods used for analysis are validated and published in the official method collection Samples analysed for presence of GMOs per year food products (15/16) feed products 632 n.d. n.d. 420 (7/16) seed lots

5 Official GMO Testing Laboratories (ENGL Members) LVUA, Neumünster LALLF MV, Rostock HU Hamburg Landeslabor BBB LAVES Braunschweig GAA Hildesheim CVUA-MEL Münster CVUA-OWL, Detmold SVUA, Arnsberg CVUA-RRW Krefeld LHL Kassel LUA Trier LUFA Speyer LSGV Saarbrücken LTZ Augustenberg CVUA Freiburg BVL BfR LVL Halle LAU Halle BfUL Radebeul LUA Dresden TLLV(LM) B.L salza TLLV(GenT) B.L salza TLL Jena LGL Oberschleißheim

6 Approaches to test for presence of GM material DNA-based analysis (PCR) targeting the genetic modification Screening 5 flank Prom enh intron gene sequence term UTR 3 flank plasmid element-specific construct-specific Detection Identification Unauthorised GMO Specificity event-specific Quantification GMO placed on market

7 Screening Official control laboratories mainly perform screening as first GMO test more than 90% of the GMO analyses are screening PCRs commonly used targets are the CaMV 35S promoter (P-35S) and the nos terminator (T-nos) P-35S T-nos present in at least 56 events present in at least 37 events BUT: If a sample is positive for 35S and/or T-nos, it may be necessary to perform all the event-specific tests to identify the GMO

8 Additional Screening Targets Screening for 35S promoter and nos terminator alone is not sufficient GMOs without P-35S or T-nos (more confidence that the analysis will detect the GM material, if present) information on the presence of other genetic elements or constructs allows the analyst to reduce the number of tests required for GMO identification Search for additional screening targets and identify genetic elements or constructs frequently present in GMOs

9 Screening Table List of all events for which sufficient information is available about all elements and genes present in the GMO data sources: CERA GM-Crop Database BATS Report EU-Register of GM Food & Feed GMDD NCBI GMDD

10 Building the Screening Table Tabulate only the important and relevant food crops maize, soya, rice, rapeseed, potato, papaya, tomato, sugarbeet, linseed, etc. Selected targets elements, genes which are frequently present Selected target for screening real-time PCR method GMOs that contain the target P-35S Yes (duplex) 56 T-nos Yes (duplex) 37 bar Yes 15 ctp2-cp4epsps Yes 12 P-35S - pat Yes 14

11 In-house Validation Validation of PCR screening methods (at single laboratory, in-house) to verify that the method is fit-for-purpose Specificity/Selectivity (positive signal only with target analyte) Sensitivity (limit of detection) Robustness (effects of small changes) theoretical verification using target sequence for computer-assisted (BLAST) searches in databases (GenBank, GMDD, CCSIS) experimental verification using (certified) reference materials or other control samples (if available)

12 Target Sequences

13 Specificity

14 Method Validation in Collaborative Trials German Working Group on GM Food Scope establish validated methods for the official collection of methods according to German Food & Feed law (Art. 64 LFGB) since 2005 focus on real-time PCR based GMO screening methods and its validation in collaborative trials fit-for-purpose, transferability Validation in CollaborativeTrials (12 16 Laboratories) DNA samples (extracted from RMs of 2 plant species) 2 levels (0,1% and 0,02%) blind-triplicate samples 6 non-gm samples false-negative / false-positive rates precision data (RSD R )

15 15 laboratories (different equipment: ABI, Mx3005, LC, Rotorgene, icycler etc.) 18 samples 3x 0,1 % LL62 DNA (20 ng/µl) 3x 0,02 % LL62 DNA (20 ng/µl) 3x 0,1 % MS8 DNA (40 ng/µl) 3x 0,02 % MS8 DNA (40 ng/µl) 3x non-gm rice (20 ng/µl) 3x non-gm rapeseed (40 ng/µl) DNA for calibration 5% LL62 DNA to prepare dilution series Example for collaborative trial Collab Validation Study of bar real-time PCR method (quantitative) Results of the collaborative trial study (2008) Number of samples per laboratory 18 Number of accepted results 240 Number of samples containing bar 180 Number of non-gm samples 60 False-positive results 0 (0 %) False-negative results 0 (0 %) 60 bp PCR product 5 flank PsuAra CTP Tg 7 3 flank UTR bar

16 Example for collaborative trial bar real-time PCR method Results of quantitations of DNA samples with 0,02% and 0,1% (cp/cp) LL62 rice and MS8 rapeseed samples GM content in % (cp/cp) Positive results calculated bar - copies % bar gene (relative to species copy number) 3 mean RSD R (%) mean 0,1 % LL62 45/ ,11 0,02 % LL62 45/ ,02 0,1 % MS8 45/ ,11 0,02 % MS8 45/ ,03 non-gm rice 0/ non-gm canola 0/ Detection limit (LOD) of bar real-time PCR about 5 copies, shown by the positive amplification in all labs using a diluted standard DNA (LL62)

17 Methods submitted to ISO TC34 SC16 ISO 24276:2006 Foodstuffs -- Methods of analysis for the detection of genetically modified organisms and derived products -- General requirements and definitions ISO 21569:2005 Foodstuffs -- Methods of analysis for the detection of genetically modified organisms and derived products -- Qualitative nucleic acid based methods ISO 21570:2005 Foodstuffs -- Methods of analysis for the detection of genetically modified organisms and derived products -- Quantitative nucleic acid based methods ISO 21571:2005 Foodstuffs -- Methods of analysis for the detection of genetically modified organisms and derived products -- Nucleic acid extraction

18 Screening Table Details X1 = authorized X2 = pending - = not authorized S = data from sequence information (e.g. plasmid map) R = analytical verification with reference material Name of Event Plant Authorization EU P35S T-nos CTP2- CP4EPSPS bar 35S-pat CRM S R S R S R S R S R soybean x 2 - wp - wp x soybean x 2 (+) wp - wp x A , A , A (LibertyLink) soybean x x A (LibertyLink) soybean wp + + x G94-1, G94-19, G-168 (Optimum) soybean GTS (Roundup Ready) soybean x x GU262 (LibertyLink) soybean MON soybean x W62, W98 (Liberty Link) soybean Microsoft Office xcel Arbeits Comment: here: information from application (FDA, 2007): parts of P35S included wp = unexpected signal, weak positive (Ct = 35 and higher)

19 Screening table use in daily work Official control laboratories perform PCR based analysis using the screening table for their daily work T-nos, P-35S, bar, 35S-pat, ctp2-epsps five targets detect most if not all events e.g. listed in CERA DB (>70 events) exceptions maize event LY038 soy event DP , MON 87701, MON 87769, BPS-CV127-9 five single real-time PCRs tests are necessary (less, if multiplex PCR is used) screening for five (or more) elements reduces number of possible events that have to be considered for further identification

20 stepwise screening maize sample positive 1st screening P-35S, T-nos, LY038 2nd screening bar, ctp2-cp4epsps, 35S-pat negative event-specific tests positive negative quantitative PCR authorised GMO unauthorised (known) GMO unauthorised (unknown) GMO GMO not detected

21 Screening table Example Maize sample: P-35S and T-nos positive / CTP2-EPSPS, bar and pat negative Name of Event Plant Authorization EU P35S T-nos CTP2- CP4EPSPS bar 35S-pat CRM 3272 maize x wp x GA 21 (Roundup Ready) maize x wp - - x LY038 maize x MIR604 maize x x MON 810 maize x x MON 863 (YieldGard) maize x x MON89034 maize x DP maize possible presence of 7 events significant difference of CT-values for the detected elements (e.g. more than 3) indicate that more than 1 event is present Microsoft Office xcel Arbeits

22 Screening table current work establish a network of sponsor laboratories sponsors are responsible for experimental verification of screening tests for a particular plant species (positive / negative reactivity) for new events that contain or not contain the target sponsors retrieve material and provide it to the network via NRL BVL performs theoretical specificity tests periodic update of table Plant Species (number of events) Lab 1 Lab 2 Lab 3 potato (8) BfUL Leipzig LALLF M-V LAVES LI-BS maize (30) IHU Hamburg CVUA-MEL NRL-GVO papaya (2) LGL Bayern rapeseed (12) CVUA FR CVUA-OWL rice (5) LHL Hessen LAV ST soybeans (10) CVUA FR NRL-GVO tomato (5) sugarbeet (3) BfUL Leipzig CVUA-OWL cotton (5) linseed (1) BfUL Leipzig

23 Screening table current work develop and validate additional screening targets P-nos, P-nos nptii, T-E9, 35S-nptII, cry1ab etc. develop multiplex PCR assays duplex (bar + pat), triplex (P-35S + T-nos + ctp2-cp4epsps) pentaplex (P-35S + T-nos + ctp2-cp4epsps + bar + pat) establish database and develop a web-based application to transfer data from Excel spreadsheets into db format (EUGINIUS MolReg project)

24 Screening Table Versions published version only interlaboratory validated methods included name of event plant authorizati on EU P35S T-nos CTP2- CP4EPSPS bar 35S-pat CRM S R S R S R S R S R maize x x in progress: advanced version with more targets (P-nos, P-FMV etc.) name of event plant authorizati on EU P35S T-nos CTP2- CP4EPSPS bar pat 35S-pat 35S-nptII pta29- pssuarabar 35S-bar Pnos-nptII barnase P-FMV CRM S R S R S R S R S R S R S R S R S R S R S R S R maize x x

25 Analytical and Bioanalytical Chemistry Volume 396, Number 6 (2010) pp (issue on GMO Analysis )