Report on the In-house Validation of a DNA Extraction Method from Oilseed rape Grains and Validated Method

In accordance with relevant EU legislation , Pioneer Overseas Corporation provided to the European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF) a DNA extraction method for oilseed rape grains and the relevant samples (ground oilseed rape grains). In line with its mandate , the EU RL GMFF has conducted an in-house validation of this DNA extraction method. To this end it tested the DNA extraction method on the samples provided and evaluated its performance in terms of DNA yield, integrity and quality. The in-house validation study confirmed that the method meets the method performance requirements as established by the EU-RL GMFF and the ENGL , and that it satisfies the provisions of Annex I-2.C.2 to Regulation (EC) No 641/2004. The method is therefore fit for the purpose of producing rapeseed DNA of suitable quantity and quality for subsequent PCR-based analysis. This report is published at http://gmo-crl.jrc.ec.europa.eu/StatusOfDossiers.htm.JRC.I.3-Molecular Biology and Genomic

Report on the Verification of the Performance of 1507, 59122, MON 810 and NK603 Event-specific PCR-based Methods applied to DNA extracted from Stack Maize 1507 x 59122 x MON 810 x NK603

An application was submitted by Pioneer Overseas Corporation to request the authorization of the genetically modified maize stack 1507 x 59122 x MON 810 x NK603, resistant against certain lepidopteran pests, protected against corn rootworm larvae, and glufosinate-ammonium and glyphosate tolerant, and all sub-combinations of the individual events as present in the segregating progeny, for food and feed uses, and import and processing, in accordance with articles 5 and 17 of Regulation (EC) N° 1829/2003 GM Food and GM Feed. The unique identifier assigned to 1507 x 59122 x MON 810 x NK603 maize is DAS-Ø15Ø7-1xDAS-59122-7xMON-ØØ81Ø-6xMON-ØØ6Ø3-6. The genetically modified maize line 1507 x 59122 x MON 810 x NK603 has been obtained by conventional crossing of four genetically modified single maize events: 1507, 59122, MON 810 and NK603 without any new genetic modification. The EU-RL GMFF has previously validated, and declared fit for purpose, the detection methods for the single events 1507, 59122, MON 810 and NK603 (see: http://gmo-crl.jrc.ec.europa.eu/statusofdoss.htm). In line with the approach defined by the ENGL (http://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Analytical_methods.pdf) the EU-RL GMFF therefore has carried out only an in-house verification of the performance of each validated method when applied to DNA extracted from 1507 x 59122 x MON 810 x NK603. The herewith reported in-house verification study lead to the conclusion that the individual methods meet the ENGL performance criteria also when applied to DNA extracted from the GM maize stack 1507 x 59122 x MON 810 x NK603.JRC.I.3-Molecular Biology and Genomic

Report on the Verification of the Performance of MON 87705 and MON 89788 Event-specific PCR-based Methods applied to DNA Extracted from GM Stack Soybean MON 87705 x MON 89788

The EU-RL GMFF has previously validated individually, and declared fit for purpose, the detection methods for the single line soybean events MON 87705 and MON 89788 and has published the corresponding reports (see http://gmo-crl.jrc.ec.europa.eu/StatusOfDossiers.aspx). In line with the approach defined by the ENGL (http://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Analytical_methods.pdf) the EU-RL GMFF therefore has carried out only an in-house verification of the performance of each validated method when applied to DNA extracted from the GM stack MON 87705 x MON 89788 soybean. The hereby reported in-house verification study led to the conclusion that the individual methods meet the ENGL requirements also when applied to DNA extracted from the GM stack MON 87705 x MON 89788 soybean.JRC.I.3-Molecular Biology and Genomic

Report on the Verification of the Performance of Bt11, DAS-59122-7, MIR604, TC 1507 and GA21 Event-specific PCR-based Methods Applied to DNA Extracted from GM Stack Bt11 x DAS-59122-7 x MIR604 x TC 1507 x GA21 Maize

An application was submitted by Syngenta Crop Protection AG to request the authorisation of genetically modified stack (GM stack) Bt11 x 59122 x MIR604 x TC1507 x GA21 maize (tolerant to herbicide products containing glufosinate ammonium/glyphosate and resistant to certain lepidopteran/coleopteran pests) and all sub-combinations of the individual events as present in the segregating progeny (except for 1507 x 59122), for food and feed uses, and import and processing, in accordance with articles 5 and 17 of Regulation (EC) No 1829/2003 on GM Food and Feed. The unique identifier assigned to GM stack Bt11 x 59122 x MIR604 x TC1507 x GA21 maize is SYN-BTØ11-1 × DAS-59122-7 × SYN-IR6Ø4-5 × DAS-Ø15Ø7-1 × MON-ØØØ21-9. The GM stack Bt11 x 59122 x MIR604 x TC1507 x GA21 maize has been obtained by conventional crossing of genetically modified maize events: Bt11, 59122, MIR604, TC1507 and GA21, without any new genetic modification. The European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF) has previously validated individually, and declared fit for purpose, the detection methods for the single events Bt11, 59122, MIR604, TC1507 and GA21 (see http://gmo-crl.jrc.ec.europa.eu/StatusOfDossiers.aspx). In line with the approach defined by the European Network of GMO Laboratories (ENGL) (http://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Analytical_methods.pdf) the EU-RL GMFF has carried out only an in-house verification of the performance of each validated method when applied to genomic DNA extracted from GM stack Bt11 x 59122 x MIR604 x TC1507 x GA21 maize. The results of the in-house verification led to the conclusion that the individual methods meet the ENGL performance criteria also when applied to genomic DNA extracted from the GM stack Bt11 x 59122 x MIR604 x TC1507 x GA21 maize.JRC.I.3-Molecular Biology and Genomic

Technical Note on the quality of DNA sequencing for the molecular characterisation of genetically modified plants

As part of the risk assessment (RA) requirements for genetically modified (GM) plants, according to Regulation (EU) No 503/2013 and the EFSA guidance on the RA of food and feed from GM plants (EFSA GMO Panel 2011), applicants need to perform a molecular characterisation of the DNA sequences inserted in the GM plant genome. This Technical Note to the applicants puts together requirements and recommendations for the quality assessment of the methodology, analysis and reporting when DNA sequencing is used for the molecular characterisation of GM plants. In particular, it applies to the use of Sanger sequencing and next-generation sequencing for the characterisation of the inserted genetic material and its flanking regions at each insertion site, the determination of the copy number of all detectable inserts and the analysis of the genetic stability of the inserts. This updated document replaces the EFSA 2018 Technical Note and reflects the current knowledge in scientific-technical methods for generating and verifying, in a standardised manner, DNA sequencing data in the context of RA of GM plants. It does not take into consideration the verification and validation of the detection method which remains under the remit of the Joint Research Centre (JRC)

Event-specific method for the quantification of oilseed rape DP-073496-4 using real-time PCR: Validation report and validated method

In line with its mandate , the European Union Reference Laboratory for GM Food and Feed (EU RL GMFF), in collaboration with the European Network of GMO Laboratories (ENGL), has validated an event-specific polymerase chain reaction (PCR) method for detecting and quantifying the oilseed rape event DP-073496-4 (unique identifier DP Ø73496-4). The validation study was conducted according to the EU-RL GMFF validation procedure [http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm] and internationally accepted guidelines (1, 2). In accordance with current EU legislation , Pioneer Overseas Corporation provided the detection method and the samples (genomic DNA extracted from oilseed rape seeds harbouring the DP-073496-4 event as positive control DNA, genomic DNA extracted from conventional oilseed rape seeds as negative control DNA). The EU-RL GMFF prepared the validation samples (calibration samples and blind samples at test GM percentage [DNA/DNA]), organised an international collaborative study and analysed the results. The study confirms that the method meets the method performance requirements as established by the EU-RL GMFF and the ENGL, in line with the provisions of Annex I-2.C.2 to Regulation (EC) No 641/2004 and it fulfils the analytical requirements of Regulation (EU) No 619/2011 .JRC.I.3-Molecular Biology and Genomic

Report on the Verification of the Performance of MON 87708 and MON 89788 Event-specific PCR-based Methods Applied to DNA Extracted from GM Stack MON 87708 x MON 89788 Soybean

An application was submitted by Monsanto Company to request the authorisation of genetically modified stack (GM stack) MON 87708 × MON 89788 soybean (tolerant to dicamba and to glyphosate) and all sub-combinations of the individual events as present in the segregating progeny, for food and feed uses, and import and processing, in accordance with articles 5 and 17 of Regulation (EC) N° 1829/2003 GM Food and GM Feed. The unique identifier assigned to GM stack MON 87708 × MON 89788 soybean is MON-877Ø8-9 x MON-89788-1. The GM stack MON 87708 × MON 89788 soybean has been obtained by conventional crossing between two genetically modified soybean events: MON 87708 and MON 89788, without any new genetic modification. The EU-RL GMFF has previously validated individually, and declared fit for purpose, the detection methods for the single events MON 87708 and MON 89788 (see http://gmo-crl.jrc.ec.europa.eu/StatusOfDossiers.aspx). In line with the approach defined by the ENGL (http://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Analytical_methods.pdf) the EU-RL GMFF has carried out only an in-house verification of the performance of each validated method when applied to genomic DNA extracted from GM stack MON 87708 × MON 89788 soybean. The results of the in-house verification led to the conclusion that the individual methods meet the ENGL performance criteria also when applied to genomic DNA extracted from the GM stack MON 87708 × MON 89788 soybean.JRC.I.3-Molecular Biology and Genomic

Identification of single target taxon-specific reference assays for the most commonly genetically transformed crops using digital droplet PCR

Knowledge of the number of DNA sequences targeted by the taxon-specific reference assays is essential for correct GM quantification and is key to the harmonisation of measurement results. In the present study droplet digital PCR (ddPCR) was used to determine the number of DNA target copies of taxon-specific assays validated for real-time PCR for the four main genetically modified (GM) crops. The transferability of experimental conditions from real-time PCR to ddPCR was also explored, as well as the effect of DNA digestion. The results of this study indicate that for each crop at least one taxon-specific assay can be identified as having a single DNA target. A short list of taxon-specific reference assays is proposed as best candidates for the relative quantification of GM events for soybean, maize, cotton and oilseed rape. The investigated assays could be in most cases transferred to ddPCR without further optimisation. The use of DNA digestion did not improve ddPCR characteristics such as rain and resolution at the conditions tested.JRC.F.5-Food and Feed Complianc

Explanatory Note - Challenges for the detection of genetically modified food or feed originating from genome editing

The recent ruling of the European Court of Justice has confirmed that organisms obtained by mutagenesis techniques are genetically modified organisms (GMOs). However, in contrast to organisms originating from conventional mutagenesis techniques, those obtained by new mutagenesis techniques are not exempted from the obligations of the GMO EU regulatory framework. This ruling raises questions about the detectability of the corresponding GM food and feed products. A case study is used in this document to explain and discuss possibilities and limitations for the detection and quantification of (known and unknown) genetic modifications in plant products derived from new mutagenesis techniques. Many of the mutations induced by new mutagenesis techniques cannot be unequivocally distinguished from natural mutations because such genome editing technologies are able to create very precise and limited genome changes that mimic the result of potential naturally occurring mutations. Moreover, mutations obtained by genome editing technologies could also not be differentiated from those introduced by conventional mutagenesis techniques which have been incorporated in traditional breeding programs and are often not thoroughly documented. Products of genome editing could only be detected and identified in imports of commodity products by enforcement laboratories when prior knowledge on the altered genome sequence, a validated detection method with appropriate selectivity and certified reference materials are available, similarly as required for the authorisation of current transgenic GMOs. However, when the modification involves only a SNP or few nucleotide changes, it would not be possible to identify whether the mutation originated spontaneously or was induced by conventional or new (genome editing) mutagenesis techniques. Moreover, it is unlikely that methods for the quantification of GMO products with small genome modifications in complex food or feed materials provide the level of selectivity needed for the enforcement of legislation, such as the one on labelling. In the absence of prior knowledge on the genome-edited changes, it is likely that non-authorised genetically modified food and feed products obtained by genome editing would enter the EU market undetected. The EU control system for GMOs and corresponding food and feed products may not function as efficiently for unauthorised genome-edited products compared to transgenic GMOs. In particular, the principle of zero tolerance for unauthorised GMO on the EU market is more difficult to maintain.JRC.F.5-Food and Feed Complianc

International Ring Trial for the Validation of an Event-Specific Golden Rice 2 Quantitative Real-Time Polymerase Chain Reaction Method

This article describes the international validation of the quantitative real-time polymerase chain reaction (PCR) detection method for Golden Rice 2. The method consists of a taxon-specific assay amplifying a fragment of rice Phospholipase D α2 gene, and an event-specific assay designed on the 3′ junction between transgenic insert and plant DNA. We validated the two assays independently, with absolute quantification, and in combination, with relative quantification, on DNA samples prepared in haploid genome equivalents. We assessed trueness, precision, efficiency, and linearity of the two assays, and the results demonstrate that both the assays independently assessed and the entire method fulfill European and international requirements for methods for genetically modified organism (GMO) testing, within the dynamic range tested. The homogeneity of the results of the collaborative trial between Europe and Asia is a good indicator of the robustness of the method.JRC.I.3-Molecular Biology and Genomic