Report on the use of EU Reference Methods and JRC decision tools for GMO analysis

To ensure harmonised scientific and technical approaches for GMO detection the European Union Reference Laboratory for GM Food and Feed (EURL GMFF) at the Joint Research Centre (JRC) has developed a freely accessible database, called “GMOMETHODS" providing a state-of-the-art catalogue of EU reference methods for GMO analysis. The EURL GMFF launched in 2015 a survey to assess the use of these EU reference methods by the official GMO control laboratories in the EU and to collect information on non-EU reference methods possibly employed for the same purpose. The survey aimed also to verify if, and to which extent, laboratories use two decision supporting tools, the JRC GMO-Matrix and Event-Finder which are available on the web site of the EURL GMFF. The survey was also directed to verify the types and frequencies of modifications possibly implemented in the protocols of the validated methods used by the official control laboratories. Results from the survey indicate that almost all official control laboratories (98 %) are using event-specific EU reference methods for quantifying GMOs while a lower number of laboratories is using EU reference methods for qualitative analyses (55 % for element-specific methods and 40 % for construct-specific methods). The use of qualitative non-EU reference methods for screening purposes may reflect the laboratory needs when facing rapid alert emergencies of quickly implementing analytical strategies for detecting non-authorised GM events. Indeed genetically modified crops have continued to increase globally, both in terms of approval status and event/trait diversification. In those cases methods validated in collaborative studies and having the status of EU-reference methods are generally not yet available. In the survey close to half of the respondents (41 %-47 %) declared also to employ to different extents the two JRC decision supporting tools, GMO-Matrix and Event-Finder. Interestingly the survey shows that almost half of the protocols of the reference methods used by the laboratories are somewhat adapted to laboratory specific conditions, mainly with respect to the master mix and the reaction volume of the polymerase chain reactions (PCR) while the primers and probes are never modified. In all cases, the impact of these modifications had been verified by the control laboratory to ensure the equivalence between the adapted and the original protocols. Without such proof, the laboratory would lose its mandatory accreditation. Moreover, participants in Comparative Testing schemes have achieve generally high score performance using those adapted methods suggesting that the modifications implemented do not affect analytical sensitivity, trueness and precision of the original protocols. The outcome of the 2015 survey reveals therefore that the combined efforts of the EURL GMFF and ENGL have been successful for enhancing harmonisation in quantitative GMO analysis by the adoption of scientific and technical approaches. This achievement allows the consistency of results for GM labelling and an equal-level playing field in the EU Member States.JRC.F.5-Food and Feed Complianc

Literature and Bioinformatics Analyses of Wheat-specific Detection Methods

In view of the recent necessity to perform testing for the detection of genetically modified common wheat (Triticum aestivum), the need arises for a taxon-specific method for this organism. However, no such method has yet been officially validated. Multiple species of wheat exist on the market, such as common wheat, durum wheat, emmer wheat, etc. These plants have complex genomes, composed of different combinations (from diploid to hexaploid) of common sets of chromosomes. The specificity of a method then depends on which set of chromosome the targeted region is located, which increases the complexity of identifying methods specific to Triticum aestivum. Often, such methods were developed for the specific regulatory need of differentiating durum and common wheat (for example, in alimentary pasta labeling), with minimal concerns for non-specific detection of other plants. This document summarises the review performed by the EU-RL GMFF, complemented with in-house bioinformatics analyses, in order to identify and characterise Triticum aestivum-specific detection methods that have been described in the scientific literature. Methods with apparent specificity (based on results shown and bioinformatics analyses) and promising performance (based on results shown) are highlighted and their primers and probe sequences reported. Those methods are the 'SS II-D' and ' SS II ex7' methods described in Matsuoka et al. (2012) and the 'wx012' method described in Iida et al. (2005), and they represent good candidates to uniquely identify common wheat in complex food samples.JRC.I.3-Molecular Biology and Genomic

Borexino : geo-neutrino measurement at Gran Sasso, Italy

Geo-neutrinos, electron anti-neutrinos produced in beta-decays of naturally occurring radioactive isotopes in the Earth, are a unique direct probe of our planet's interior. After a brief introduction of the geo-neutrinos' properties and of the main aims of their study, we discuss the features of a detector which has recently provided breakthrough achievements in the field, Borexino, a massive, calorimetric liquid scintillator detector installed at the underground Gran Sasso Laboratory. With its unprecedented radiopurity levels achieved in the core of the detection medium, it is the only experiment in operation able to study in real time solar neutrino interactions in the challenging sub-MeV energy region. Its superior technical properties allowed Borexino also to provide a clean detection of terrestrial neutrinos. Therefore, the description of the characteristics of the detected geo-neutrino signal and of the corresponding geological implications are the main core of the discussion contained in this work

article ID bav101; V C The Author(s) Database

These authors contributed equally to this work. Abstract The DNA target sequence is the key element in designing detection methods for genetically modified organisms (GMOs). Unfortunately this information is frequently lacking, especially for unauthorized GMOs. In addition, patent sequences are generally poorly annotated, buried in complex and extensive documentation and hard to link to the corresponding GM event. Here, we present the JRC GMO-Amplicons, a database of amplicons collected by screening public nucleotide sequence databanks by in silico determination of PCR amplification with reference methods for GMO analysis. The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) provides these methods in the GMOMETHODS database to support enforcement of EU legislation and GM food/feed control. The JRC GMO-Amplicons database is composed of more than 240 000 amplicons, which can be easily accessed and screened through a web interface. To our knowledge, this is the first attempt at pooling and collecting publicly available sequences related to GMOs in food and feed. The JRC GMOAmplicons supports control laboratories in the design and assessment of GMO methods, providing inter-alia in silico prediction of primers specificity and GM targets coverage. The new tool can assist the laboratories in the analysis of complex issues, such as the detection and identification of unauthorized GMOs. Notably, the JRC GMO-Amplicons database allows the retrieval and characterization of GMO-related sequences included in patents documentation. Finally, it can help annotating poorly described GM sequences and identifying new relevant GMO-related sequences in public databases. The JRC GMO-Amplicons is freely accessible through a web-based portal that is hosted on the EU-RL GMFF website

GMOMETHODS: The European Union Database of Reference Methods for GMO Analysis

In order to provide reliable and harmonized information on methods for GMO (genetically modified organism) analysis we have published a database called “GMOMETHODS” that supplies information on PCR assays validated according to the principles and requirements of ISO 5725 and/or the International Union of Pure and Applied Chemistry protocol. In addition, the database contains methods that have been verified by the European Union Reference Laboratory for Genetically Modified Food and Feed in the context of compliance with an European Union legislative act. The web application provides search capabilities to retrieve primers and probes sequence information on the available methods. It further supplies core data required by analytical labs to carry out GM tests and comprises information on the applied reference material and plasmid standards. The GMOMETHODS database currently contains 118 different PCR methods allowing identification of 51 single GM events and 18 taxon-specific genes in a sample. It also provides screening assays for detection of eight different genetic elements commonly used for the development of GMOs. The application is referred to by the Biosafety Clearing House, a global mechanism set up by the Cartagena Protocol on Biosafety to facilitate the exchange of information on Living Modified Organisms. The publication of the GMOMETHODS database can be considered an important step toward worldwide standardization and harmonization in GMO analysis.JRC.I.3-Molecular Biology and Genomic

JRC GMO-Amplicons, a collection of nucleic acid sequences related to genetically modified organisms

The DNA target sequence is the key element in designing detection methods for genetically modified organisms (GMOs). Unfortunately this information is frequently lacking, especially for unauthorised GMOs. In addition, patent sequences are generally poorly annotated, buried in complex and extensive documentation and hardly associable to the corresponding GM event. Here we present the JRC GMO-Amplicons, a database of amplicons collected by screening public nucleotide sequence databanks by in silico determination of PCR amplification with reference methods of GMO analysis. The European Union Reference Laboratory for Genetically Modified Food and Feed of the European Commission (EU-RL GMFF) provides these methods in the GMOMETHODS database for supporting enforcement of EU legislation and GM food/feed control. The JRC GMO-Amplicons database is composed of more than 240,000 amplicons, which can be easily accessed and screened through a web-interface. To our knowledge, this is the first attempt of pooling and collecting publicly available sequences related to GMOs in food and feed. The JRC GMO-Amplicons supports control laboratories in the design and assessment of GMO methods, providing inter-alia in silico prediction of primers specificity and GM targets coverage. The new tool may assist the laboratories in the analysis of complex issues, such as detection and identification of unauthorised GMOs. Notably, the JRC GMO-Amplicons database may allow the retrieval and characterisation of GMO-related sequences also included in patents documentation. Finally, it could help annotating poorly described GM sequences and identifying new relevant GMO-related sequences in public databases. The JRC GMO-Amplicons is freely accessible through a web-based portal that is hosted on the EURL-GMFF web-site.JRC.I.3-Molecular Biology and Genomic

JRC GMO-Matrix: a web application to support Genetically Modified Organisms detection strategies

Background: The polymerase chain reaction (PCR) is the current state of the art technique for DNA-based detection of Genetically Modified Organisms (GMOs). A typical control strategy starts by analyzing a sample for the presence of target sequences (GM-elements) known to be present in many GMOs. Positive findings from this “screening” are then confirmed with GM (event) specific test methods. A reliable knowledge of which GMOs are detected by combination of GM-detection methods is thus crucial to minimize the verification efforts. Description: In this article, we describe a novel platform that links the information of two unique databases built and maintained by the European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) at the Joint Research Centre (JRC) of the European Commission, one containing the sequence information of known GM-events and the other validated PCR-based detection and identification methods. The new platform compiles in silico determinations of the detection of a wide range of GMOs by the available detection methods using existing scripts that simulate PCR amplification and, when present, probe binding. The correctness of the information has been verified by comparing the in silico conclusions to experimental results for a subset of forty-nine GM events and six methods. Conclusions: The JRC GMO-Matrix is unique for its reliance on DNA sequence data and its flexibility in integrating novel GMOs and new detection methods. Users can mine the database using a set of web interfaces that thus provide a valuable support to GMO control laboratories in planning and evaluating their GMO screening strategies. The platform is accessible at http://gmo-crl.jrc.ec.europa.eu/jrcgmomatrix/JRC.I.3-Molecular Biology and Genomic

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

In vivo functional analysis of the Ras exchange factor son of sevenless

The Son of sevenless (Sos) protein functions as a guanine nucleotide transfer factor for Ras and interacts with the receptor tyrosine kinase Sevenless through the protein Drk, a homolog of mammalian Grb2. In vivo structure-function analysis revealed that the amino terminus of Sos was essential for its function in flies. A molecule lacking the amino terminus was a potent dominant negative. In contrast, a Sos fragment lacking the Drk binding sites was functional and its activity was dependent on the presence of the Sevenless receptor. Furthermore, membrane localization of Sos was independent of Drk. A possible role for Drk as an activator of Sos is discussed and a Drk-independent interaction between Sos and Sevenless is proposed that is likely mediated by the pleckstrin homology domain within the amino terminus

Functional roles for the pleckstrin and Dbl homology regions in the Ras exchange factor Son-of-sevenless

Activation of p21ras by receptor tyrosine kinases is thought to result from recruitment of guanine nucleotide exchange factors such as Son-of-sevenless (Sos) to plasma membrane receptor substrates via adaptor proteins such as Grb2. This hypothesis was tested in the present studies by evaluating the ability of truncation and deletion mutants of Drosophila (d)Sos to enhance [32P]GTP loading of p21ras when expressed in 32P-labeled COS or 293 cells. The dSos catalytic domain (residues 758-1125), expressed without the dSos NH2-terminal (residues 1-757) or adaptor-binding COOH-terminal (residues 1126-1596) regions, exhibits intrinsic exchange activity as evidenced by its rescue of mutant Saccharomyces cerevisiae deficient in endogenous GTP/GDP exchange activity. Here we show that this dSos catalytic domain fails to affect GTP p21ras levels when expressed in cultured mammalian cells unless the NH2-terminal domain is also present. Surprisingly, the COOH-terminal, adaptor binding domain of dSos was not sufficient to confer p21ras exchange activity to the Sos catalytic domain in these cells in the absence of the NH2-terminal domain. This function of promoting catalytic domain activity could be localized by mutational analysis to the pleckstrin and Dbl homology sequences located just NH2-terminal to the catalytic domain. The results demonstrate a functional role for these pleckstrin and Dbl domains within the dSos protein, and suggest the presence of unidentified cellular elements that interact with these domains and participate in the regulation of p21ras