Event-specific Method for the Quantification of Sugar beet line H7-1 Using Real-time PCR v. 1.01

Transcription

1 Event-specific Method for the Quantification of Sugar beet line H7-1 Using Real-time PCR v Validation Report and Validated Method Marco Mazzara N. Foti C. Savini G. Van den Eede 2013 Report EUR EN

2 European Commission Joint Research Centre Institute for Health and Consumer Protection Contact information Molecular Biology and Genomics Unit Address: Joint Research Centre, Via Enrico Fermi 2749, TP 201, Ispra (VA), Italy Tel.: Fax: Legal Notice Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use which might be made of this publication. Europe Direct is a service to help you find answers to your questions about the European Union Freephone number (*): (*) Certain mobile telephone operators do not allow access to numbers or these calls may be billed. A great deal of additional information on the European Union is available on the Internet. It can be accessed through the Europa server This document replaces Event Event-specific Method for the Quantification of Sugar beet line H7-1 1 Using Real-time PCR- Validation Report and Protocol with ISBN number and PUBSY request number JRC The corrections made in the new document are: Validation Report Page 7 5: For specific detection of event H7-1 genomic DNA, a 110-bp fragment changed by For specific detection of event H7-1 genomic DNA, a 108-bp fragment For relative quantitation of event H7-1 DNA, a sugar beet reference system amplifies a 121-bp fragment changed by For relative quantitation of event H7-1 DNA, a sugar beet reference system amplifies a 118-bp fragment Validated method Page 4 1: For specific detection of event H7-1 genomic DNA, a 110-bp fragment changed by For specific detection of event H7-1 genomic DNA, a 108-bp fragment For relative quantitation of event H7-1 DNA, a sugar beet reference system amplifies a 121-bp fragment changed by For relative quantitation of event H7-1 DNA, a sugar beet reference system amplifies a 118-bp fragment JRC84121 EUR EN ISBN ISSN doi: /21128 Luxembourg: Publications Office of the European Union, 2013 European Union, 2013 Reproduction is authorised provided the source is acknowledged. Printed in Italy

3 CRLVL28/04VR corrected version 2 EUROPEAN COMMISSION JOINT RESEARCH CENTRE Institute for Health and Consumer Protection Molecular Biology and Genomics Unit Event-specific method for the quantitation of sugar beet line H7-1 using real-time PCR Validation Report Joint Research Centre Institute for Health and Consumer Protection Unit for Molecular Biology and Genomics 31 January 2006 Corrected version 1-19/05/2008 (see page 2) Corrected version 2-28/08/2013 (see page 2) Executive Summary The JRC as European Union Reference Laboratory (EURL) for the GM Food and Feed (see Regulation EC 1829/2003), in collaboration with the European Network of GMO Laboratories (ENGL), has carried out a collaborative study to assess the performance of a quantitative event-specific method to detect and quantify the H7-1 transformation event in sugarbeet DNA (unique identifier KM-ØØØH71-4). The collaborative trial was conducted according to internationally accepted guidelines. Monsanto provided the method-specific samples (seeds of sugarbeet line H7-1, 100% event H7-1 and seeds of non-gm conventional sugarbeet line, 0% event H7-1) whereas the JRC, upon extraction of the respective DNA, prepared the validation samples (calibration samples and blind samples at unknown GM percentage). The trial involved thirteen laboratories from ten European countries. The results of the collaborative trial fully met ENGL s performance requirements and the scientific understanding about satisfactory method performance. Therefore, the JRC as European Union Reference Laboratory considers the method validated as fit for the purpose of regulatory compliance. The results of the collaborative study are publicly available under The method will also be submitted to CEN, the European Standardisation body, to be considered as international standard. European Union Reference Laboratory for GM Food and Feed 1/13 Validation Report Sugar beet H7-1

4 CRLVL28/04VR corrected version 2 Quality assurance The EU-RL GMFF is ISO 17025:2005 accredited [certificate number: ACCREDIA 1172, (Flexible Scope for DNA extraction and qualitative/quantitative PCR) - Accredited tests are available at The original version of the document containing evidence of internal checks and authorisation for publication is archived within the EU-RL GMFF quality system. The EU-RL GMFF is also ISO 17043:2010 accredited (Proficiency test provider) and apply the corresponding procedures and processes for the management of ring trials during the method validation. The EU-RL GMFF conducts its activities under the certification ISO 9001:2008 of the IHCP Institute provided by CERMET Correction from the previous versions: Corrected version 1-19/05/2008 Page 7 5: For specific detection of event H7-1 genomic DNA, a 110-bp fragment changed by For specific detection of event H7-1 genomic DNA, a 108-bp fragment Corrected version 2-28/08/2013 Page 7 5: For relative quantitation of event H7-1 DNA, a sugar beet reference system amplifies a 121-bp fragment changed by For relative quantitation of event H7-1 DNA, a sugar beet reference system amplifies a 118-bp fragment Address of contact laboratory: European Commission, Joint Research Centre (JRC) Institute for Health and Consumer Protection (IHCP) Unit for Molecular Biology and Genomics European Union Reference Laboratory for GM Food and Feed Via E. Fermi 2749, Ispra (VA) Italy Functional mailbox: eurl-gmff@jrc.ec.europa.eu European Union Reference Laboratory for GM Food and Feed Validation Report Sugar beet H7-1 2/13

5 CRLVL28/04VR corrected version 2 Contents 1. INTRODUCTION LIST OF PARTICIPANTS MATERIALS EXPERIMENTAL DESIGN METHOD... 7 DESCRIPTION OF THE OPERATIONAL STEPS DEVIATIONS REPORTED SUMMARY OF RESULTS... 8 PCR EFFICIENCY AND LINEARITY... 8 GMO QUANTITATION METHOD PERFORMANCE REQUIREMENTS SPECIFICITY OF THE GS (GLUTAMINE SYNTHETASE) REFERENCE GENE CONCLUSIONS REFERENCES European Union Reference Laboratory for GM Food and Feed Validation Report Sugar beet H7-1 3/13

6 CRLVL28/04VR corrected version 2 1. Introduction The Joint Research Centre (JRC, Biotechnology and GMOs Unit of the Institute of Health and Consumer Protection) as European Union Reference Laboratory for the GM Food and Feed (see Regulation EC 1829/2003) organised the collaborative trial of the event-specific method for the detection and quantification of H7-1 sugarbeet. The study involved thirteen laboratories, members of the European Network of GMO Laboratories (ENGL). Upon reception of methods, samples and related data, the JRC carried out the scientific evaluation of documentation and the in-house testing of the methods, according to the requirements of Regulation (EC) 641/2004 and following its operational procedures. The internal tests were carried out in March-May Following the evaluation of the data and the results of the laboratory tests, the ring trial was organized and took place in June A method for DNA extraction from sugarbeet seeds, submitted by the applicant, was evaluated by the JRC; laboratory testing of the method was carried out in order to confirm its performance. The protocol was employed for the extraction of DNA samples used in this validation study. The protocol for DNA extraction and a report on method testing is available under The operational procedure of the collaborative study comprised the following module: Quantitative real-time PCR (Polymerase Chain Reaction). The methodology is an eventspecific real-time quantitative TaqMan PCR procedure for the determination of the relative content of event H7-1 DNA to total sugarbeet DNA. The procedure is a simplex system, in which a sugarbeet GS (glutamine synthetase) endogenous assay (reference gene) and the target assay (H7-1) are performed in separate wells. The PCR assay has been optimised for use in real-time PCR instruments for plastic reaction vessels. The ring-trial was carried out in accordance with the following internationally accepted guidelines: ISO 5725 (1994). The IUPAC Protocol for the design, conduct and interpretation of methodperformance studies (Horwitz, 1995). European Union Reference Laboratory for GM Food and Feed Validation Report Sugar beet H7-1 4/13

7 CRLVL28/04VR corrected version 2 2. List of Participants The method was tested in thirteen ENGL laboratories to determine its performance. Each laboratory was requested to carefully follow the protocol provided. The participating laboratories are listed in Table 1 in alphabetical order. Table 1. ENGL laboratories participating in the validation study of sugar beet H7-1. Laboratory Bundesinstitut fuer Risikobewertung (BfR) CRA-W, Dépt Qualité des productions agricoles Danish Institute for Food and Veterinary Research Danish Plant Directorate Ente Nazionale Sementi Elette Istituto Zooprofilattico Sperimentale Lazio e Toscana Laboratoire MDO Unité PMDV Laboratoire National de Santé LAV Sachsen-Anhalt National Food Administration National Institute of Food Hygiene and Nutrition GMO lab Swiss Federal Research Station for Animal Production and Dairy Products Umweltbundesamt GmbH Country Germany Belgium Denmark Denmark Italy Italy France Luxembourg Germany Sweden Hungary Switzerland Austria European Union Reference Laboratory for GM Food and Feed Validation Report Sugar beet H7-1 5/13

8 CRLVL28/04VR corrected version 2 3. Materials For the validation of the quantitative event-specific method, seeds from sugarbeet line RM- HybH7-A constituted the positive control for event H7-1 and conventional sugarbeet seeds from line RM-HybConv-B were the negative control. Following DNA extraction, samples containing mixtures of 0% and 100% H7-1 sugarbeet genomic DNA at different GMO concentrations were prepared by the JRC. The participants received the following materials: Four calibration samples (180 µl of DNA solution each) labelled from S1 to S4. Twenty unknown DNA samples (90 µl of DNA solution each), labelled from U1 to U20. Amplification reagent control was used on each PCR plate. Reaction reagents as follows: PCR buffer I 10X, one tube : 1.5 ml Rox Reference Dye (25M), one tube: 500 l MgCl 2 (25mM), two tubes: 1.7 ml each datp (10mM), one tube: 320 l dctp (10mM), one tube: 320 l dgtp (10mM), one tube: 320 l dutp (20mM), one tube: 320 l Ampli Taq Gold (5U/l), one tube: 112 l Distilled sterile water, one tube: 5 ml Primers and probes (1 tube each) as follows: GS system GluA3-F primer (100 µm): 11 µl GluA3-R (100 µm): 11 µl GluD1TaqMan probe (100 µm): 7 µl H7-1 sugar beet system H7PLT1 primer Forward. (100 µm): 28 µl ZRH7-R2 primer Reverse (100 µm): 28 µl ZRH7-prob1 TaqMan probe (100 µm): 7 µl European Union Reference Laboratory for GM Food and Feed Validation Report Sugar beet H7-1 6/13

9 CRLVL28/04VR corrected version 2 Table 2 shows the GM contents of the unknown samples over the dynamic range. Table 2. H7-1 GM contents H7-1 GM % [GM copy number/sugar beet genome copy number (*100)] Experimental design Twenty unknown samples (ten for each of two plates), representing five GM levels, were used in the validation study. On each PCR plate, samples were analyzed in parallel with both the H7-1 and GS gene specific system. Two plates in total were run. Each GM level was quantified in quadruplicate and each sample was analyzed in three replicates. Participating laboratories carried out the determination of the GM% according to the instructions provided in the protocol and using the electronic tool provided (Excel spreadsheet). 5. Method Description of the operational steps For specific detection of event H7-1 genomic DNA, a 108-bp fragment of the recombination region of parts of the construct inserted into the plant genome is amplified using two specific primers. PCR products are measured during each cycle (real-time) by means of a targetspecific oligonucleotide probe labelled with two fluorescent dyes: FAM as a reporter dye at its 5 end and TAMRA as a quencher dye at its 3 end. For relative quantification of event H7-1 DNA, a sugarbeet-specific reference system amplifies a 118-bp fragment of GS (glutamine synthetase) a sugar beet endogenous gene, using a pair of GS gene-specific primers and a GS gene-specific probe labelled with FAM and TAMRA. The standard curves are generated both for the GS and H7-1 specific system by plotting the Ct-values measured for the calibration points against the logarithm of the DNA copy numbers, and by fitting a linear regression line into these data. Thereafter, the standard curves are used to estimate the copy numbers in the unknown sample DNA by interpolation from the standard curves. For the determination of the amount of H7-1 DNA in the unknown sample, the H7-1 copy number is divided by the copy number of the sugarbeet reference gene (GS) and multiplied by 100 to obtain the percentage value (GM% = H7-1/GS * 100). European Union Reference Laboratory for GM Food and Feed Validation Report Sugar beet H7-1 7/13

10 CRLVL28/04VR corrected version 2 The calibration sample S1 is was derived from a 10% H7-1 sample, prepared by mixing the appropriate amount of H7-1 DNA from the stock solution in control non-gm sugar beet DNA. This corresponds to 10,000 GM copies in 125 ng of DNA. The absolute copy numbers in the calibration curve samples are determined by dividing the sample DNA weight (nanograms) by the published average 1C value for sugar beet genome (1.25 pg) (Arumuganathan & Earle, 1991). The copy number values, which were used in the quantification, are provided in Table 3. Table 3. Copy number values of the standard curve samples. Sample code S1 S2 S3 S4 Total amount (ng) of DNA in reaction Sugarbeet genome copies 100,000 20,000 4, H7-1 GM copies 10,000 2, Deviations reported Seven laboratories reported no deviations from the protocol. The volume for the master mix was calculated for the effective number of wells to be loaded, without any extra-volume to account for random error pipetting. As a consequence, two laboratories prepared extra-tubes of master mix to replenish the control samples. One laboratory reduced by 7% the volume of master mix and DNA to be added to each well. One laboratory performed the runs a second time after modifying the master mix volume but lost a sample corresponding to one GM-level. One laboratory performed twice the same run due to an erroneous manipulation of one plate during a first loading. One laboratory had problems with the optical cover and lost a few repetitions of the S-samples (4 in total) without affecting the quality of the standard-curves. 7. Summary of results PCR efficiency and linearity The values of the slopes [from which the PCR efficiency is calculated using the formula ((10^(- 1/slope))-1)*100] of the standard curves and of the R 2 (expressing the linearity of the regression) reported by participating laboratories for both PCR systems and runs (reference gene and GM specific, plate A and B), are summarised in Table 4. European Union Reference Laboratory for GM Food and Feed Validation Report Sugar beet H7-1 8/13

11 CRLVL28/04VR corrected version 2 Table 4. Values of standard curve slope, PCR efficiency and linearity (R 2 ) for the reference gene (GS) and the GM specific (H7-1) systems H7-1 GS LAB PLATE Slope PCR Efficiency (%) Linearity (R 2 ) Slope PCR Efficiency (%) Linearity (R 2 ) 1 A B A B A B A B A B A B A B A B A B A B A B A B A B Mean European Union Reference Laboratory for GM Food and Feed Validation Report Sugar beet H7-1 9/13

12 CRLVL28/04VR corrected version 2 Data reported in Table 4 confirm the performance characteristics of the method tested. The mean PCR efficiency for the event-specific system was 90%, while that of the endogenous reference-system was 93.34%. Two laboratories reported important deviations from the mean of the remaining laboratories. When those data were not taken into account, the mean of PCR efficiency in the H7-1 system rose to 94.65% and that of the GS system remained basically unchanged (93.9%). The linearity of the method was very good being 0.98 in the H7-1 system and almost 1.00 in the GS system. Again, after eliminating the deviating values reported by two laboratories, the mean of the linearity in the H7-1 system increased to GMO quantitation Table 5 shows the mean values of the four repetitions for each GM level as provided by all laboratories. Each mean value is the average of three PCR replicates. Table 5. Replicates mean value by laboratories and by all unknown samples. GMO content of samples (GM% = GM copy number/sugar beet genome copy number *100) LAB 0.1% 0.5% 0.9% 2.0% 5.0% REP 1 REP 2 REP 3 REP 4 REP 1 REP 2 REP 3 REP 4 REP 1 REP 2 REP 3 REP 4 REP 1 REP 2 REP 3 REP 4 REP 1 REP 2 REP 3 REP In Figure 1 the relative deviation from the true value for each GM level and per laboratory is shown. The coloured bars represent the relative GM quantification obtained in the participating laboratories; the red bar represents the overall mean. In this computation, the values of two laboratories for each GM-level were excluded since they were identified as outliers following Cochrane and Grubbs analysis. The mean bias of the GM quantitation was modest at each GMlevel, indicating that the good correlation between estimated and true value. Only one laboratory showed a deviation from the true value greater than 25% at the 0.90% level. No overall overestimation/underestimation trend can be observed. European Union Reference Laboratory for GM Food and Feed Validation Report Sugar beet H7-1 10/13

13 CRLVL28/04VR corrected version 2 Figure 1. Relative deviation (%) from the true value of H7-1 for all laboratories 8. Method performance requirements The results of the collaborative trial are reported in table 6. These are evaluated with respect to the method acceptance criteria and to the method performance requirements, as established by ENGL and adopted by EURL GMFF. In table 6 estimates of both repeatability and reproducibility for each GM level are reported, after identification and removal of outliers through Cochran and Grubbs tests, according to ISO Table 6. Sugar beet H7-1 validation data. Expected value (GMO %) Unknown sample GM% Laboratories having returned results Samples per laboratory Number of outliers Reason for exclusion 2C 2C 1C, 1G 1C, 1G 1C 1G Mean value Repeatability relative standard deviation (%) Repeatability standard deviation Reproducibility relative standard deviation (%) Reproducibility standard deviation Bias (absolute value) Bias (%) C. test = Cochran s test; G. test = Grubbs test European Union Reference Laboratory for GM Food and Feed Validation Report Sugar beet H7-1 11/13

14 CRLVL28/04VR corrected version 2 The relative reproducibility standard deviation (RSD R ), that describes the inter-laboratory variation, should be below 33% at the target concentration and over the majority of the dynamic range, while it should be below 50% at the lower end of the dynamic range. As it can be observed in table 6, the method fully satisfies this requirement at all GM level tested. In fact, the highest value of RSD R (%) is at the 0.10% level, well within the acceptance criterion. In the same table the relative repeatability standard deviation (RSD r ) values are also reported, as estimated from ring trial results for each GM level. In order to accept methods for collaborative trial evaluation, the EURL GMFF requires that RSD r is below 25%, as indicated by ENGL. As it can be observed from the values reported in table 6, the method satisfies this requirement throughout the whole dynamic range tested. In table 6 measures of method bias, which allow estimating trueness, are also shown for each GM level. Bias is estimated according to ISO 5725 data analysis protocol. According to ENGL method performance requirements, trueness should be 25% throughout the whole dynamic range. In this case the method excellently satisfies such requirement throughout the whole dynamic range tested. 9. Specificity of the GS (glutamine synthetase) reference gene Experimental tests conducted by the applicant have shown that the GS reference system cannot discriminate between sugar beet (Beta vulgaris) and autumn beet (Brassica rapa). Out of the six autumn beet varieties, chosen to represent the genetic variability of Brassica rapa, two reacted with the sugar beet reference system. However, in these cases, Ct figures of autumn beet, in subsequent tests carried out by the applicant upon request of the EURL GMFF, differ significantly from those obtained from samples of sugar beet when the same amount of DNA was analyzed, corresponding to a Brassica rapa reactivity from two thousand to at least thirty-two thousand times less than that of Beta vulgaris. 10. Conclusions The overall method performance has been evaluated with respect to the method acceptance criteria and method performance requirements recommended by the ENGL (available under The method acceptance criteria were reported by the applicant and used to evaluate the method prior to the collaborative study. The results obtained during the collaborative trial indicate that the method can be considered as fit for enforcement purposes with respect to its intra- and inter-laboratory variability, trueness and with the observations on the reference system reported above. European Union Reference Laboratory for GM Food and Feed Validation Report Sugar beet H7-1 12/13

15 CRLVL28/04VR corrected version References Arumuganathan, K., Earle, E.D. (1991). Nuclear content of some important plant species. Plant Mol Biol Reporter 9, Horwitz, W. (1995) Protocol for the design, conduct and interpretation of method performance studies, Pure and Appl. Chem, 67, International Standard (ISO) Accuracy (trueness and precision) of measurement methods and results. International Organization for Standardization, Genève, Swizerland. European Union Reference Laboratory for GM Food and Feed Validation Report Sugar beet H7-1 13/13

16 CRLVL28/04VP corrected version 2 Annex 1: Event-specific method for the quantitation of sugar beet line H7-1 using real-time PCR Validated method 31/01/2006 Corrected version 1-19/05/2008 (see page 2) Corrected version 2-28/08/2013 (see page 2) Method development: KWS SAAT AG and Monsanto Company Method validation: Joint Research Centre European Commission Unit for Molecular Biology and Genomics 1/11 -

17 Protocol H7-1 European Union Reference Laboratory for GM Food and Feed CRLVL28/04VP corrected version 2 Quality assurance The EU-RL GMFF is ISO 17025:2005 accredited [certificate number: ACCREDIA 1172, (Flexible Scope for DNA extraction and qualitative/quantitative PCR) - Accredited tests are available at The original version of the document containing evidence of internal checks and authorisation for publication is archived within the EU-RL GMFF quality system. The EU-RL GMFF is also ISO 17043:2010 accredited (Proficiency test provider) and apply the corresponding procedures and processes for the management of ring trials during the method validation. The EU-RL GMFF conducts its activities under the certification ISO 9001:2008 of the IHCP Institute provided by CERMET Correction from the previous versions: Corrected version 1-19/05/2008 Page 4 1: For specific detection of event H7-1 genomic DNA, a 110-bp fragment changed by For specific detection of event H7-1 genomic DNA, a 108-bp fragment Corrected version 2-28/08/2013 Page 4 1: For relative quantitation of event H7-1 DNA, a sugar beet reference system amplifies a 121-bp fragment changed by For relative quantitation of event H7-1 DNA, a sugar beet reference system amplifies a 118-bp fragment Address of contact laboratory: European Commission, Joint Research Centre (JRC) Institute for Health and Consumer Protection (IHCP) Unit for Molecular Biology and Genomics European Union Reference Laboratory for GM Food and Feed Via E. Fermi 2749, Ispra (VA) Italy Functional mailbox: eurl-gmff@jrc.ec.europa.eu 2/11

18 Protocol H7-1 European Union Reference Laboratory for GM Food and Feed CRLVL28/04VP corrected version 2 Contents 1. GENERAL INFORMATION AND SUMMARY OF THE METHODOLOGY VALIDATION STATUS AND PERFORMANCE CHARACTERISTICS GENERAL COLLABORATIVE TRIAL LIMIT OF DETECTION LIMIT OF QUANTITATION MOLECULAR SPECIFICITY PROCEDURES GENERAL INSTRUCTIONS AND PRECAUTIONS REAL-TIME PCR FOR QUANTITATIVE ANALYSIS OF H7-1 SUGARBEET General Calibration Real-time PCR set-up DATA ANALYSIS CALCULATION OF RESULTS MATERIALS EQUIPMENT REAGENTS PRIMERS AND PROBES REFERENCES /11

19 Protocol H7-1 European Union Reference Laboratory for GM Food and Feed CRLVL28/04VP corrected version 2 1. General information and summary of the methodology This protocol describes an event-specific real-time quantitative TaqMan PCR procedure for the determination of the relative content of event H7-1 DNA to total sugarbeet DNA in a sample. The PCR assay has been optimised for use in real-time PCR instruments for plastic reaction vessels. Glass capillaries are not recommended for the buffer composition described in this method. Template DNA extracted by means of suitable methods should be tested for quality and quantity prior to the use in PCR assay. Tests for the presence of PCR inhibitors (e.g. monitor run, use of DNA spikes) are recommended. For specific detection of event H7-1 genomic DNA, a 108-bp fragment of the recombination region of parts of the construct inserted into the plant genome is amplified using two specific primers. PCR products are measured during each cycle (real-time) by means of a target-specific oligonucleotide probe labelled with two fluorescent dyes: FAM as a reporter dye at its 5 end and TAMRA as a quencher dye at its 3 end. For relative quantitation of event H7-1 DNA, a sugar beet reference system amplifies a 118-bp fragment of the glutamine synthetase (GS) gene, a sugarbeet endogenous gene, using a pair of GS gene-specific primers and a GS gene-specific probe labelled with FAM and TAMRA as described above. The measured fluorescence signal passes a threshold value after a certain number of cycles. This threshold cycle is called the Ct value. For quantitation of the amount of event H7-1 DNA in a test sample, event H7-1 and GS Ct values are determined for the sample. Standard curves are then used to calculate the relative content of event H7-1 DNA to total maize DNA. 2. Validation status and performance characteristics 2.1 General The method has been optimised for DNA extracted from seeds, containing mixtures of genetically modified H7-1 and conventional sugar beet. The reproducibility and trueness of the method was tested through collaborative trial using samples at different GMO contents. 4/11

20 Protocol H7-1 European Union Reference Laboratory for GM Food and Feed CRLVL28/04VP corrected version Collaborative trial The method was validated in a collaborative trial by the Joint Research Centre (JRC) of the European Commission. The study was undertaken with thirteen laboratories. Each participant received twenty unknown samples containing H7-1 sugar beet genomic DNA at five concentration levels, between 0.1 % and 5.0 %. Each test sample was analyzed by PCR in three repetitions. The study was designed as a blind quadruplicate collaborative trial; each laboratory received each level of GM H7-1 in four unknown samples. Two replicates of each GM level were analyzed on the same PCR plate. A detailed validation report can be found under Limit of detection According to the method developer, the absolute LOD of the method is 10 copies. The relative LOD was not assessed in a collaborative trial. The lowest relative concentration of the target sequence included in collaborative trial was 0.10%. 2.4 Limit of quantitation According to the method developer, the relative LOQ of the method is 0.045%. The lowest relative concentration of the target sequence included in collaborative trial was 0.10%. 2.5 Molecular specificity The method utilizes a unique DNA sequence of the recombination region of parts of the construct inserted into the plant genome. The sequence is specific to H7-1 and thus imparts event-specificity to the detection method. The specificity was assessed by Blastsearch at the National Center for Biotechnology Information (NCBI) with the Standard nucleotide-nucleotide BLAST [blastn] ( on the amplicon resulting from the eventspecific amplification of the transition region of the sugar beet genomic DNA into the specific event. No 100% match with other plant GMO sequences was found. The specificity was experimentally tested against DNA extracted from plant materials containing the specific targets (at least 500 genomic copies/reaction) of Bt176 Corn, NK603 Corn, Mon810 Corn, Bt11 Corn, GA21 RR-Corn, T25 Corn, Mon863 Corn, RR- Soybean, RR Rapeseed, and against DNA from non-gm sources, i.e. rice, wheat, spinach, potato, chard, beetroot, autumn beet and sugarbeet. 5/11

21 Protocol H7-1 European Union Reference Laboratory for GM Food and Feed CRLVL28/04VP corrected version 2 None of the materials yielded detectable amplification. Experimental tests conducted by the applicant have shown that the GS reference system cannot discriminate between sugar beet (Beta vulgaris) and autumn beet (Brassica rapa). Out of the six autumn beet varieties, chosen to represent the genetic variability of Brassica rapa, two reacted with the sugar beet reference system. However, in such cases, Ct figures of autumn beet, in subsequent tests carried out by the applicant upon request of the EURL GMFF, differ significantly from those obtained from samples of sugar beet when the same amount of DNA was analyzed, corresponding to a Brassica rapa reactivity from two thousand to at least thirty-two thousand times less than that of Beta vulgaris. 3. Procedures 3.1 General instructions and precautions All handling of reagents and controls should occur in an ISO 9001:2000 or ISO environment or equivalent. The procedures require experience of working under sterile conditions. Laboratory organization, e.g. forward flow direction during PCR-setup, should follow the guidelines given by relevant authorities like e.g. ISO, CEN, Codex Alimentarius Commission. PCR-reagents shall be stored and handled in a separate room and freezer and in equipment where no nucleic acids (with exception of PCR primers or probes) or DNA degrading or modifying enzymes have been handled previously. All handling of PCR reagents and controls requires dedicated equipment especially pipettes. All the equipment used must be sterilized prior to use and any residue of DNA has to be removed. All material used (e.g. vials, containers, pipette tips, etc.) must be suitable for PCR and molecular biology applications. They must be DNase-free, DNA-free, sterile and shall not adsorb protein or DNA. In order to avoid contamination, filter pipette tips protected against aerosol should be used. Use only powder-free gloves and change them frequently. Clean lab-benches and equipment periodically with 10% sodium hypochloride solution (bleach). Pipettes should be checked regularly for precision and calibrated, if necessary. 6/11

22 Protocol H7-1 European Union Reference Laboratory for GM Food and Feed CRLVL28/04VP corrected version 2 All handling steps - unless specified otherwise - shall be carried out at 0-4 C. In order to avoid repeated freeze/thaw cycles, aliquots should be prepared. 3.2 Real-time PCR for quantitative analysis of H7-1 sugarbeet General The PCR set-up for the taxon specific target sequence (GS) and for the GMO (H7-1) target sequence should be carried out in separate vials. Multiplex PCR (using differential fluorescent labels for the probes) has not been tested or validated. The use of maximum 125 ng of template DNA per reaction well is recommended. The method is developed for a total volume of 25 µl per reaction mixture with the reagents as listed in Table 1 and Table Calibration Separate calibration curves with each primer/probe system are generated in the same analytical amplification run. The calibration curves consist of four samples. The first point of the calibration curves is a 10% H7-1 in non-gm sugar beet DNA for a total of 125 ng of DNA per reaction (corresponding to 100,000 sugar beet genome copies with one genome assumed to correlate to 1.25 pg of haploid sugarbeet genomic DNA) (Arumuganathan & Earle, 1991). A series of 1:5 dilutions (starting from the first point) down to 1 ng of total sugarbeet DNA per reaction may be used. A calibration curve is produced by plotting Ct-values against the logarithm of the target copy number for the calibration points. This can be done e.g. by use of spreadsheet software, e.g. Microsoft Excel, or directly by options available with the sequence detection system software. The copy numbers measured for the unknown sample DNA is obtained by interpolation from the standard curves Real-time PCR set-up 1. Thaw, mix gently and centrifuge the required amount of components needed for the run. Keep thawed reagents at 1-4 C on ice. 7/11

23 Protocol H7-1 European Union Reference Laboratory for GM Food and Feed CRLVL28/04VP corrected version 2 2. In two reaction tubes (one for H7-1 system and one for the GS system) on ice, add the following components (Tables 1 and 2) in the order mentioned below (except DNA) to prepare the master mixes. Table 1. Amplification reaction mixture in the final volume/concentration per reaction well for the reference GS specific system. Component Final concentration µl/reaction PCR buffer I 10x Rox Reference Dye (25M) MgCl 2 (25mM) datps (10mM) dctps (10mM) dgtps (10mM) dutps (20mM) GluA3-F primer (100M) GluA3-R primer (100M) GluD1 probe (100M) Ampli Taq Gold (5U/l) Nuclease free water 1x 1 M 5 mm 0.2 mm 0.2 mm 0.2 mm 0.4 mm 150 nm 150 nm 100 nm 0.04 U/l # Template DNA (see and 3.2.2) Total reaction volume: 25 Table 2. Amplification reaction mixture in the final volume/concentration per reaction well for H7-1 specific system. Component Final concentration µl/reaction PCR buffer I 10x Rox Reference Dye (25M) MgCl 2 (25mM) datps (10mM) dctps (10mM) dgtps (10mM) dutps (20mM) H7PLT1 primer (100M) 1x 1 M 7 mm 0.2 mm 0.2 mm 0.2 mm 0.4 mm 400 nm 400 nm /11

24 Protocol H7-1 European Union Reference Laboratory for GM Food and Feed CRLVL28/04VP corrected version 2 ZRH7-R2 primer (100M) ZRH7 probe (100M) Ampli Taq Gold (5U/l) 100 nm 0.04 U/l # Nuclease free water Template DNA (see and 3.2.2) Total reaction volume: 25 9/11

25 CRLVL28/04VP corrected version 2 3. Mix gently and centrifuge briefly. 4. Prepare two reaction tubes (one for the H7-1 and one for the GS master mix) for each DNA sample to be tested (reference curve samples, unknown samples and control samples). 5. Add to each reaction tube the correct amount of master mix (e.g. 20 x 3 = 60 µl master mix for three PCR repetitions). Add to each tube the correct amount of DNA (e.g. 5 x 3 = 15 µl DNA for three PCR repetitions). Low-speed vortex each tubes at least three times for approx 30 sec. This step is mandatory to reduce the variability among the repetitions of each sample to a minimum. 6. Spin down the tubes in a micro-centrifuge. Aliquot 25 µl in each well. Seal the reaction plate with optical cover or optical caps. Centrifuge the plate at low speed (e.g. approximately 250 x g for 1 minute at 4 C to room temperature) to spin down the reaction mixture. 7. Place the plate into the instrument. 8. Run the PCR with cycling conditions described in Table 3: Table 3. Reaction conditions. Step Stage T C Time (sec) Acquisition Cycles 1 Initial denaturation 95 C 600 No 1x 2a Denaturation 95 o C 15 No 2b Amplification Annealing & 60 o C 60 Measure 45x Extension 3.3 Data analysis Subsequent to the real-time PCR, analyse the run following the procedure below: a) Set the threshold: display the amplification curves of one system (e.g. H7-1) in logarithmic mode. Locate the threshold line in the area where the amplification profiles are parallel (exponential phase of PCR) and where there is no fork effect between repetitions of the same sample. Press the update button to ensure changes affect Ct values. Switch to the linear view mode by clicking on the Y axis of the amplification plot, and check that the threshold previously set falls within the geometric phase of the curves. 10/11 -

26 Protocol H7-1 European Union Reference Laboratory for GM Food and Feed CRLVL28/04VP corrected version 2 b) Set the baseline: determine the cycle number at which the threshold line crosses the first amplification curve and set the baseline three cycles before that value (e.g. earliest Ct = 25, set the baseline crossing at Ct = 25 3 = 22). c) Save the settings d) Repeat the procedure described in a) and b) on the amplification plots of the other system (e.g. GS system). e) Save the settings and export all the data into an Excel file for further calculations. 3.4 Calculation of results After having defined a threshold value within the logarithmic phase of amplification as described above, the instruments software calculated the Ct-values for each reaction. The standard curves are generated both for the GS and H7-1 specific system by plotting the Ct-values measured for the calibration points against the logarithm of the DNA copy numbers, and by fitting a linear regression line into these data. Thereafter, the standard curves are used to estimate the copy numbers in the unknown sample DNA by interpolation from the standard curves. For the determination of the amount of H7-1 DNA in the unknown sample, the H7-1 copy number is divided by the copy number of the sugarbeet reference gene (GS) and multiplied by 100 to obtain the percentage value (GM% = H7-1/GS * 100). 4. Materials 4.1 Equipment Real-time PCR instrument for plastic reaction vessels (glass capillaries are not recommended for the described buffer composition) Plastic reaction vessels suitable for real-time PCR instrument (enabling undisturbed fluorescence detection) Software for evaluating data after standard curve method (mostly integrated in the software of the real-time PCR instrument) Microcentrifuge Micropipettes Vortex Rack for reaction tubes 1.5/2.0 ml tubes 11/11

27 Protocol H7-1 European Union Reference Laboratory for GM Food and Feed CRLVL28/04VP corrected version 2 Reagents (equivalents may be substituted) PCR buffer I 10x (Applied Biosystems Part No. N ) MgCl 2 (Applied Biosystems Part No. N ) Rox (Invitrogen PartNo ) datp (Amersham-Pharmacia Part No ) dctp (Amersham-Pharmacia Part No ) dgtp (Amersham-Pharmacia Part No ) dutp (Amersham-Pharmacia Part No ) ATGold (Applied Biosystems Part No. N ) TE-Buffer ph=8.0 (10/1 mm) (Applichem Part No. A2575,1000) Primers and Probes Name Oligonucleotide DNA Sequence (5 to 3 ) H7-1 target sequence H7PLT1 5 - TGG GAT CTG GGT GGC TCT AAC T -3 ZRH7-R2 5 - AAT GCT GCT AAA TCC TGA G -3 ZRH7 (Probe) FAM-5 - AAG GCG GGA AAC GAC AAT CT -3 -TAMRA Reference gene GS target sequence GluA3-F 5 - GAC CTC CAT ATT ACT GAA AGG AAG -3 GluA3-R 5 - GAG TAA TTG CTC CAT CCT GTT CA -3 GluD1 (Probe) FAM-5 - CTA CGA AGT TTA AAG TAT GTG CCG CTC -3 -TAMRA 5. References Arumuganathan, K., Earle, E.D. (1991). Nuclear content of some important plant species. Plant Mol Biol Reporter 9, /11

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29 How to obtain EU publications Our priced publications are available from EU Bookshop ( where you can place an order with the sales agent of your choice. The Publications Office has a worldwide network of sales agents. You can obtain their contact details by sending a fax to (352) European Commission EUR EN Joint Research Centre Institute for Health and Consumer Protection Title: Event-Specific Method for the Quantitation of Sugarbeet Line H7-1 Using Real-Time PCR - Validation Report corr. version 2 and Validated Method corr. version 2 Author(s): Marco Mazzara, N. Foti, C. Savini, G. Van den Eede Luxembourg: Publications Office of the European Union pp x 29.7 cm EUR Scientific and Technical Research series ISSN ISBN doi: /21128 Abstract The JRC as European Union Reference Laboratory (EURL) for the GM Food and Feed (see Regulation EC 1829/2003), in collaboration with the European Network of GMO Laboratories (ENGL), has carried out a collaborative study to assess the performance of a quantitative event-specific method to detect and quantify the H7-1 transformation event in sugarbeet DNA (unique identifier KM-ØØØH71-4). The collaborative trial was conducted according to internationally accepted guidelines. Monsanto provided the method-specific samples (seeds of sugarbeet line H7-1, 100% event H7-1 and seeds of non-gm conventional sugarbeet line, 0% event H7-1) whereas the JRC, upon extraction of the respective DNA, prepared the validation samples (calibration samples and blind samples at unknown GM percentage). The trial involved thirteen laboratories from ten European countries. The results of the collaborative trial fully met ENGL's performance requirements and the scientific understanding about satisfactory method performance. Therefore, the JRC as Community Reference Laboratory considers the method validated as fit for the purpose of regulatory compliance. The results of the collaborative study are publicly available under The method will also be submitted to CEN, the European Standardisation body, to be considered as international standard.

30 LB-NA EN-N As the Commission s in-house science service, the Joint Research Centre s mission is to provide EU policies with independent, evidence-based scientific and technical support throughout the whole policy cycle. Working in close cooperation with policy Directorates-General, the JRC addresses key societal challenges while stimulating innovation through developing new methods, tools and standards, and sharing its know-how with the Member States, the scientific community and international partners. Key policy areas include: environment and climate change; energy and transport; agriculture and food security; health and consumer protection; information society and digital agenda; safety and security, including nuclear; all supported through a cross-cutting and multi-disciplinary approach. doi: /21128