Report on the Single-laboratory Validation of a PCR-based Detection Method for Identification of Florigene™ 26407 GM Carnation

Suntory Holdings Ltd has submitted an application for marketing (C/NL/09/02) of a genetically modified carnation line 26407 (Unique identifier: IFD-26407-2). In this context, the European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF) was asked to carry out a singlelaboratory validation of the performance of a polymerase chain reaction (PCR)-based method for detecting and identifying the carnation GM line 26407, developed by the applicant. This report describes the results of this validation, carried out by the EU-RL GMFF with control samples provided by the applicant. The method is a duplex end-point PCR, where a carnation (taxon) target and a transgenic sequence are detected simultaneously. The limit of detection (LOD) of the method has been established to be at least 10 copies for the taxon-specific target and between 50 and 10 copies for the GM target, based on haploid genome copy number. The event-specificity of the method was assessed by the applicant as being sufficient. The EU-RL could verify that the taxon-specific primers correctly detect the endogenous gene target in genomic DNA of a conventional carnation line (negative control) and in the genomic DNA of the GM carnation line, while the GM target is detected by the GM specific primers only in genomic DNA of 26407 GM line (positive control).JRC.I.3-Molecular Biology and Genomic

Event-specific Method for the Quantification of Soybean CV127 using Real-time PCR

The European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF), established by Regulation (EC) No 1829/2003, has carried out a validation study to assess the performance of a quantitative event-specific method on the soybean event CV127 (unique identifier BPS-CV127-9). The collaborative trial was conducted according to internationally accepted guidelines. In accordance to Regulation (EC) No 1829/2003 of 22 September 2003 on genetically modified food and feed and to Regulation (EC) No 641/2004 of 6 April 2004 on detailed rules for the implementation of Regulation (EC) No 1829/2003, BASF Plant Science provided the detection method and the control samples. The EU-RL GMFF prepared the validation samples [calibration samples and blind samples at unknown GM percentages (DNA/DNA)].The results of the international collaborative trial met the European Network of GMO Laboratories (ENGL) method performance requirements (http://gmocrl.jrc.ec.europa.eu/guidancedocs.htm). The method is therefore considered applicable to the control samples provided, in accordance with the requirements of Annex I – 2.C.2 to Regulation (EC) No 641/2004.JRC.I.3-Molecular Biology and Genomic

Event-specific Method for the Quantification of Maize MON87460 Using Real-time PCR: Validation Report and Validated Method

The European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF), established by Regulation (EC) No 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 MON 87460 transformation event in maize DNA (unique identifier MON-8746Ø-4). The collaborative study was conducted according to internationally accepted guidelines (1, 2). In accordance with Regulation (EC) No 1829/2003 of 22 September 2003 on genetically modified food and feed and with Regulation (EC) No 641/2004 of 6 April 2004 on detailed rules for the implementation of Regulation (EC) No 1829/2003, Monsanto Company provided the detection method and the samples (genomic DNA extracted from homogenised seeds containing the transformation event and from conventional homogenised seeds). The EU-RL GMFF prepared the validation samples (calibration samples and blind samples at defined GM percentages [DNA/DNA], unknown to laboratories participating to the collaborative study). The collaborative trial involved twelve laboratories from ten European countries. The results of the international collaborative study met the ENGL performance requirements. The method is, therefore, considered applicable to the control samples provided, in accordance with the requirements of Annex I-2.C.2 to Commission Regulation (EC) No 641/2004. The results of the collaborative study are made publicly available at http://gmo-crl.jrc.ec.europa.eu/.JRC.I.3-Molecular Biology and Genomic

Report on the Verification of the Performance of MON 88017 and MON 810 Event-specific Methods on the Maize Event MON 88017 x MON 810 Using Real-Time PCR

The JRC as Community Reference Laboratory for GM Food and Feed (CRL-GMFF), established by Regulation (EC) No 1829/2003, has carried out an in-house verification study to assess the performance of two quantitative event-specific methods on the maize event MON 88017 x MON 810 (unique identifier MON-88Ø17-3 x MON-ØØ810-6) which combines the MON 88017 and MON 810 transformation events. The two methods have been previously validated individually on single-trait event, to detect and quantify each event in maize samples; a validation report for each method is available at http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm. This study was conducted according to internationally accepted guidelines (1, 2). In accordance to Regulation (EC) No 1829/2003 of 22 September 2003 on genetically modified food and feed and to Regulation (EC) No 641/2004 of 6 April 2004 on detailed rules for the implementation of Regulation (EC) No 1829/2003, Monsanto Europe S.A. provided the detection methods and the control samples: whole seeds of MON 88017 x MON 810 maize (TPX151-DT, GLP-0409-15526-S) and whole conventional maize seeds of EXP258 (GLP-0409-15528-S). The JRC prepared the in-house verification samples (calibration samples and blind samples at different GM percentages). The results of the in-house verification study were evaluated with reference to ENGL method performance requirements (http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm) and to the validation results on the individual parental events (http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm). The results of this CRL-GMFF in-house verification studies are made publicly available at http://gmo-crl.jrc.ec.europa.eu/.JRC.DDG.I.4-Molecular biology and genomic

In-house validation of an Event-specific Method for the Quantification of Oliseed Rape RF2 using Real-time PCR

The European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF), established by Regulation (EC) No 1829/2003, has carried out an in-house validation study to assess the performance of a quantitative event-specific method on the oilseed rape event RF2 (unique identifier ACS-BN002-5). In accordance to Regulation (EC) No 1829/2003 of 22 September 2003 on genetically modified food and feed and to Regulation (EC) No 641/2004 of 6 April 2004 on detailed rules for the implementation of Regulation (EC) No 1829/2003, Bayer CropScience provided the detection method and the control samples. The EU-RL GMFF prepared the validation samples [calibration samples and blind samples at different GM percentages (DNA/DNA)]. The results of the in-house validation were evaluated with respect to method acceptance criteria and method performance requirements recommended by the European Network of GMO Laboratories (ENGL) (http://gmocrl.jrc.ec.europa.eu/guidancedocs.htm) and to its applicability in different real-time PCR instruments. The results obtained indicate that the method complies with the ENGL criteria. The method is therefore considered applicable to the control samples provided, in accordance with the requirements of Annex I – 2.C.2 to Regulation (EC) No 641/2004.JRC.I.3-Molecular Biology and Genomic

Event-specific Method for the Quantification of Soybean MON87769 Using Real-time PCR: Validation Report and Validated Method

The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF), established by Regulation (EC) No 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 MON87769 transformation event in soybean DNA (unique identifier MON-87769-7). The collaborative trial was conducted according to internationally accepted guidelines (1, 2). In accordance to Regulation (EC) No 1829/2003 of 22 September 2003 “on genetically modified food and feed” and to Regulation (EC) No 641/2004 of 6 April 2004 “on detailed rules for the implementation of Regulation (EC) No 1829/2003”, Monsanto provided the detection method and the control samples (genomic DNA extracted from soybean seeds harbouring the MON87769 event and from conventional soybean seeds). The EU-RL GMFF prepared the validation samples (calibration samples and samples at unknown GM percentage [DNA/DNA]). The collaborative trial involved twelve laboratories from nine European countries. The results of the international collaborative trial met the ENGL performance requirements. The method is therefore considered applicable to the control samples provided, in accordance with the requirements of Annex I-2.C.2 to Regulation (EC) No 641/2004. The results of the collaborative study are made publicly available at http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm.JRC.I.3-Molecular Biology and Genomic

Event-specific Method for the Quantification of Cotton MON 88913 Using Real-time PCR

The JRC as Community Reference Laboratory for GM Food and Feed (CRL-GMFF), established by Regulation (EC) No 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 MON 88913 transformation event in cotton DNA (unique identifier MON-88913-8). The collaborative trial was conducted according to internationally accepted guidelines (1, 2). In accordance with Regulation (EC) No 1829/2003 of 22 September 2003 ¿on genetically modified food and feed¿ and with Regulation (EC) No 641/2004 of 6 April 2004 ¿on detailed rules for the implementation of Regulation (EC) No 1829/2003¿, Monsanto provided the detection method and the samples: genomic DNA from cotton seeds harbouring the MON 88913 event (line ST 4664) and from conventional cotton seeds (line ST 474). The JRC prepared the validation samples (calibration samples and blind samples at unknown GM percentage [DNA/DNA]). The collaborative trial involved twelve laboratories from nine European countries. The results of the international collaborative trial met the ENGL performance requirements. The method is, therefore, considered applicable to the control samples provided, in accordance with the requirements of Annex I-2.C.2 to Regulation (EC) No 641/2004. The results of the collaborative study are made publicly available at http://gmo-crl.jrc.ec.europa.eu/JRC.DDG.I.4-Molecular biology and genomic

Report on the Verification of the Performance of a Construct-Specific Assay for the Detection of Flax CDC Triffid Event FP967 Using Real-Time PCR

Further to the detection by the German authorities of the unauthorised flax CDC Triffid event FP967 (Unique Identifier CDC-FLØØ1-2) in materials imported from Canada, a notification was sent through the Rapid Alert System for Food and Feed (RASFF) in September 2009. On 21st August 2009, the Community Reference Laboratory for Genetically Modified Food and Feed received from the German authorities a construct-specific method for the detection of flax CDC Triffid event FP967, developed by Genetic ID, Augsburg (Germany). The method developer declared this method as specific for event FP967 as it targets a transition sequence spanning the nopaline synthase (nos) terminator gene and the spectinomycin/streptomycin resistance gene, construction being found only in the flax FP967 event. On 11th September 2009, the CRL-GMFF received from the German authorities the FP967 positive control in the form of DNA extracted from seeds. Seeds were provided to the German authorities by the University of California, Riverside, USA. The CRL-GMFF carried out experiments on the control sample received in order to verify the specificity and the Limit of Detection (LOD) of the construct-specific method. The CRL-GMFF observed that the NOST-Spec (nos terminator ¿ spectinomycin resistance gene) construct-specific method generates a PCR amplification product of 105 bp, whose sequence is homologous to a transition sequence spanning the nopaline synthase (nos) terminator gene and the dihydrofolate reductase gene. The experimental testing of the specificity indicates that the NOST-Spec construct-specific assay does not detect genetically modified events under the conditions reported. The limit of detection (LOD) established is between 1 and 5 haploid genome copies of FP967.JRC.DDG.I.4-Molecular biology and genomic

Event-specific method for the quantification of soybean MON 87705 using real-time PCR validation report

The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF), established by Regulation (EC) No 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 MON 87705 transformation event in soybean DNA (unique identifier MON-877Ø5-6). The collaborative trial was conducted according to internationally accepted guidelines (1, 2). In accordance with Regulation (EC) No 1829/2003 of 22 September 2003 “on genetically modified food and feed” and with Regulation (EC) No 641/2004 of 6 April 2004 “on detailed rules for the implementation of Regulation (EC) No 1829/2003”, Monsanto provided the detection method and the samples (genomic DNA from soybean seeds harbouring the MON 87705 event and from conventional soybean seeds). The EU-RL GMFF prepared the validation samples (calibration samples and blind samples at unknown GM percentage [DNA/DNA]). The collaborative trial involved twelve laboratories from nine European countries. The results of the international collaborative trial met the ENGL performance requirements. The method is, therefore, considered applicable to the control samples provided, in accordance with the requirements of Annex I-2.C.2 to Regulation (EC) No 641/2004. The results of the collaborative study are made publicly available at http://gmo-crl.jrc.ec.europa.eu/JRC.I.3-Molecular Biology and Genomic

Event-specific Method for the Quantification of Soybean Line MON 89788 Using Real-time PCR v. 1.01: Validation Report and Validated Method

The JRC as European Union Reference Laboratory for GM Food and Feed (EURL-GMFF), established by Regulation (EC) No 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 MON89788 transformation event in soybean DNA (unique identifier MON-89788-1). The collaborative trial was conducted according to internationally accepted guidelines (1, 2). In accordance with Regulation (EC) No 1829/2003 of 22 September 2003 on genetically modified food and feed and with Regulation (EC) No 641/2004 of 6 April 2004 on detailed rules for the implementation of Regulation (EC) No 1829/2003, Monsanto provided the detection method and the samples (soybean seeds containing the transformation event and conventional soybean seeds). The JRC prepared the validation samples (calibration samples and blind samples at unknown GM percentage [DNA/DNA]). The collaborative trial involved twelve laboratories from eight European countries. The results of the international collaborative trial met the ENGL performance requirements. The method is therefore considered applicable to the control samples provided, in accordance with the requirements of Annex I-2.C.2 to Commission Regulation (EC) No 641/2004.JRC.I.3-Molecular Biology and Genomic