Alexandre Angers-Loustau

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

BackgroundThe 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 combinations of GM-detection methods is thus crucial to minimize the verification efforts.DescriptionIn 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.ConclusionsThe 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/.

Development and applicability of a ready-to-use PCR system for GMO screening.

With the growing number of GMOs introduced to the market, testing laboratories have seen their workload increase significantly. Ready-to-use multi-target PCR-based detection systems, such as pre-spotted plates (PSP), reduce analysis time while increasing capacity. This paper describes the development and applicability to GMO testing of a screening strategy involving a PSP and its associated web-based Decision Support System. The screening PSP was developed to detect all GMOs authorized in the EU in one single PCR experiment, through the combination of 16 validated assays. The screening strategy was successfully challenged in a wide inter-laboratory study on real-life food/feed samples. The positive outcome of this study could result in the adoption of a PSP screening strategy across the EU; a step that would increase harmonization and quality of GMO testing in the EU. Furthermore, this system could represent a model for other official control areas where high-throughput DNA-based detection systems are needed.

Baseline Practices for the Application of Genomic Data Supporting Regulatory Food Safety

The application of new data streams generated from next-generation sequencing (NGS) has been demonstrated for food microbiology, pathogen identification, and illness outbreak detection. The establishment of best practices for data integrity, reproducibility, and traceability will ensure reliable, auditable, and transparent processes underlying food microbiology risk management decisions. We outline general principles to guide the use of NGS data in support of microbiological food safety. Regulatory authorities across intra- and international jurisdictions can leverage this effort to promote the reliability, consistency, and transparency of processes used in the derivation of genomic information for regulatory food safety purposes, and to facilitate interactions and the transfer of information in the interest of public health.

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 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 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 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. Database URL: http://gmo-crl.jrc.ec.europa.eu/jrcgmoamplicons/

Novel nuclear barcode regions for the identification of flatfish species

The development of an efficient seafood traceability framework is crucial for the management of sustainable fisheries and the monitoring of potential substitution fraud across the food chain. Recent studies have shown the potential of DNA barcoding methods in this framework, with most of the efforts focusing on using mitochondrial targets such as the cytochrome oxidase 1 and cytochrome b genes. In this article, we show the identification of novel targets in the nuclear genome, and their associated primers, to be used for the efficient identification of flatfishes of the Pleuronectidae family. In addition, different in silico methods are described to generate a dataset of barcode reference sequences from the ever-growing wealth of publicly available sequence information, replacing, where possible, labour-intensive laboratory work. The short amplicon lengths render the analysis of these new barcode target regions ideally suited to next-generation sequencing techniques, allowing characterisation of multiple fish species in mixed and processed samples. Their location in the nucleus also improves currently used methods by allowing the identification of hybrid individuals.

Screening for six genetically modified soybean lines by an event-specific multiplex PCR method: Collaborative trial validation of a novel approach for GMO detection

AbstractThis study presents a novel approach to detect genetically modified (GM) plant events that are not covered by common GMO screening methods. It is based on a simplified multiplex assay which merges the event-specific real-time PCR methods for the detection of six GM soybean lines (MON 87701, MON 87708, MON 87769, DP-305423, CV-127 and DAS-68416). The use of two different fluorescent dyes facilitates the subsequent analysis for identification of the GM event. The multiplex PCR method was validated in a collaborative study trial with 16 participating laboratories. Each laboratory received eight samples containing low levels (0.1% or 0.03% m/m) of one or two GM soybean lines and four GM-negative samples. Data of 720 PCR analyses were evaluated and a false-positive rate of 0.3% and a false-negative rate of 3.9% was observed, respectively. The limits of detection (LOD 95%) were calculated based on modelling the probability of detection (POD) and show satisfactory sensitivity and reproducibility for the assay. Furthermore, we discuss the modularity and applicability of event-specific multiplex PCR systems for the detection of GM events that are not covered by screenings.

Molecular characterization of an unauthorized genetically modified Bacillus subtilis production strain identified in a vitamin B2 feed additive

Highlights • Genetically modified Bacillus subtilis identified in a vitamin B2 product.• Whole genome sequencing runs are performed for characterization of the isolated strain.• Complex modifications of the genome are identified.• Four putative recombinant plasmids are characterized.• Real-time PCR methods are developed and available for testing vitamin B2 products.

Towards Plant Species Identification in Complex Samples: A Bioinformatics Pipeline for the Identification of Novel Nuclear Barcode Candidates.

Monitoring of the food chain to fight fraud and protect consumer health relies on the availability of methods to correctly identify the species present in samples, for which DNA barcoding is a promising candidate. The nuclear genome is a rich potential source of barcode targets, but has been relatively unexploited until now. Here, we show the development and use of a bioinformatics pipeline that processes available genome sequences to automatically screen large numbers of input candidates, identifies novel nuclear barcode targets and designs associated primer pairs, according to a specific set of requirements. We applied this pipeline to identify novel barcodes for plant species, a kingdom for which the currently available solutions are known to be insufficient. We tested one of the identified primer pairs and show its capability to correctly identify the plant species in simple and complex samples, validating the output of our approach.

The European Union Reference Methods Database and Decision Supporting Tool for the Analysis of Genetically Modified Organisms: GMOMETHODS and JRC GMO-Matrix

Enforcement of European Union legislation on genetically modified organisms (GMOs) requires the availability of reliable, sensitive, specific methods and their harmonized application. The European Union Reference Laboratory for GM Food and Feed has developed a series of technical approaches to enable the implementation of an analytically demanding but operational legal framework. These include a freely accessible database called “GMOMETHODS,” for providing a state-of-the-art catalog of verified and standardized methods for GMO analysis, a dynamic matrix-based web-application called JRC GMO-Matrix for efficiently preparing and evaluating GMO screening strategies, and ready-to-use prespotted plates as a multitarget tool to drastically decrease the laboratory workload. These approaches are globally available and can be considered as an important contribution for worldwide standardization and harmonization in GMO analysis.

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.