Strategies to detect unauthorized GMO in the food and feed chain

To guarantee the traceability on the market and the freedom of choice for consumers, genetically modified organisms (GMO) legislations have been established in many countries, including in Europe (EU). However, the implementation of these legislations by the enforcement laboratories is becoming complex due mainly to the increasing number and diversity of GMO. To cope with the problematic of EU unauthorized GMO, this PhD aims to improve and strengthen the existing GMO detection system using high-tech approaches. First, as a study case, an overview of genetically modified (GM) rice, developed around the world was carried out to collect information related inter alia on elements found in their transgenic cassette. Second, according to this information, key targets, frequently found in GMO (p35S and tNOS) or exclusively observed in EU unauthorized GMO (t35S pCAMBIA), were selected to develop a strategy allowing to detect and characterize a broad range of GMO. This strategy, fully integrated in the GMO routine analysis, consists to characterize sequences surrounding detected key transgenic elements using a DNA walking approach. By this way, the acquisition of sequences from the junction between the transgenic cassette and the plant genome as well as the associations of elements typically found in transgenic constructs allow to confirm the presence of GMO in food/feed matrices. Due to its good performance thoroughly assessed via several unprocessed and processed food/feed matrices, this strategy represents a key tool, easily implementable by the enforcement laboratories. With the aim to even more simplify the workflow and increase the throughput of this strategy, the sequencing step was performed using the Next Generation Sequencing (NGS) technology instead of the Sanger technology. In parallel, the detection of GMO in alimentary matrices using exclusively the NGS technology, through a whole genome sequencing (WGS) approach, was also investigated. As this last approach does not theoretically require any prior information about the targeted sequences, GMO composed only of unknown transgenic elements could be detected. This work has thus allowed to provide additional strategies to the current GMO detection system in order to characterize a larger spectrum of GMO, both authorized or not

Integrated DNA walking system to characterize a broad spectrum of GMOs in food/feed matrices

Background: In order to provide a system fully integrated with qPCR screening, usually used in GMO routine analysis, as well as being able to detect, characterize and identify a broad spectrum of GMOs in food/feed matrices, two bidirectional DNA walking methods targeting p35S or tNOS, the most common transgenic elements found in GM crops, were developed. These newly developed DNA walking methods are completing the previously implemented DNA walking method targeting the t35S pCAMBIA element. Results: First, the newly developed DNA walking methods, anchored on the sequences used for the p35S or tNOS qPCR screening, were tested on Bt rice that contains these two transgenic elements. Second, the methods were assessed on a maize sample containing a low amount of the GM MON863 event, representing a more complex matrix in terms of genome size and sensitivity. Finally, to illustrate its applicability in GMO routine analysis by enforcement laboratories, the entire workflow of the integrated strategy, including qPCR screening to detect the potential presence of GMOs and the subsequent DNA walking methods to characterize and identify the detected GMOs, was applied on a GeMMA Scheme Proficiency Test matrix. Via the characterization of the transgene flanking region between the transgenic cassette and the plant genome as well as of a part of the transgenic cassette, the presence of GMOs was properly confirmed or infirmed in all tested samples. Conclusion: Due to their simple procedure and their short time-frame to get results, the developed DNA walking methods proposed here can be easily implemented in GMO routine analysis by the enforcement laboratories. In providing crucial information about the transgene flanking regions and/or the transgenic cassettes, this DNA walking strategy is a key molecular tool to prove the presence of GMOs in any given food/feed matrix

Strategy for the identification of micro-organisms producing food and feed products : bacteria producing food enzymes as study case

Recent European regulations require safety assessments of food enzymes (FE) before their commercialization. FE are mainly produced by micro-organisms, whose viable strains nor associated DNA can be present in the final products. Currently, no strategy targeting such impurities exists in enforcement laboratories. Therefore, a generic strategy of first line screening was developed to detect and identify, through PCR amplification and sequencing of the 16S-rRNA gene, the potential presence of FE producing bacteria in FE preparations. First, the specificity was verified using all microbial species reported to produce FE. Second, an in-house database, with 16S reference sequences from bacteria producing FE, was constructed for their fast identification through blast analysis. Third, the sensitivity was assessed on a spiked FE preparation. Finally, the applicability was verified using commercial FE preparations. Using straightforward PCR amplifications, Sanger sequencing and blast analysis, the proposed strategy was demonstrated to be convenient for implementation in enforcement laboratories

Current and new approaches in GMO detection: challenges and solutions

In many countries, genetically modified organisms (GMO) legislations have been established in order to guarantee the traceability of food/feed products on the market as well as to protect the consumer freedom of choice. Therefore, several GMO detection strategies, mainly based on DNA, have been developed to implement these legislations. Due to its numerous advantages, the quantitative PCR (qPCR) is the method of choice for the enforcement laboratories in GMO routine analysis. However, given the increasing number and diversity of GMO developed and put on the market around the world, some technical hurdles could be encountered with the qPCR technology, mainly owed to its inherent properties. To address these challenges, alternative GMO detection methods have been developed, allowing faster detections of single GM target (e.g. Loop-mediated isothermal amplification), simultaneous detections of multiple GM targets (e.g. PCR capillary gel electrophoresis, microarray and Luminex®), more accurate quantification of GM targets (e.g. digital PCR) or characterization of partially known (e.g. DNA walking and Next Generation Sequencing (NGS)) or unknown (e.g. NGS) GMO. The benefits and drawbacks of these methods are discussed in this review

ddPCR strategy to detect a gene-edited plant carrying a single variation point: Technical feasibility and interpretation issues

International audienceGene-edited organisms and derived food and feed products commercialized on the European market falls within the scope of the Directive, 2001/18/EC. Therefore, the possibility to specifically detect and quantify them has become a priority. To this end, PCR-based approaches, such as real-time PCR and digital droplet PCR, targeting a single variation point carried by a gene-edited organism are expected to be suitable, even if potentially challenging at the technical level. However, additional issues related to the interpretation of the results can also be encountered. Indeed, given its possible spread, natural or through breeding programs, the presence of this single variation does not automatically prove the presence of the gene-edited organism. To overcome such critical issue, we proposed a general workflow to develop and validate a PCR-based method specific to a gene-edited organism in targeting its single variation point. First, based on in silico analyses, the possibility to technically design the PCR-based method as well as to discriminate the gene-edited organism using it single variation point are assessed. In case such parameters are confirmed, the performance of the developed PCR-based method are then tested in agreement with the minimum performance requirements for GMO testing. The use of the proposed general workflow was successfully illustrated through the development a 2-plex digital droplet PCR method targeting specifically a gene-edited rice carrying a single nucleotide insertion. The proposed workflow was thus considered as a key tool to support the competent authorities regarding the food and feed traceability

First monitoring for unauthorized genetically modified bacteria in food enzymes from the food market

Recently, several methods were developed to detect genetically modified (GM) bacteria, allowing to screen for the potential presence of bacteria and to prove bacterial GM contaminations in fermentation products (such as FE). To obtain a first overview of the possible contamination of FE with GM bacteria, a general workflow was proposed, combining recently developed detection methods. Those methods include a 16S screening and viability assessment to target bacterial contaminations, as well as a first line and second line qPCR screening targeting bacterial GMM in FE preparations. Subsequently, the proposed workflow was applied on a total of 51 representative FE preparations that were collected from the EU market. On this basis, several FE contaminated with a bacterial GM strain, including the presence of full-length AMR genes, could be identified. In addition, the presence of two specific GMM (both viable and associated recombinant DNA) could be confirmed in multiple FE as well as the presence of viable GMM. On this basis, numerous bacterial contaminations, including GMM carrying full-length antimicrobial resistance (AMR) genes, were detected and confirmed. The generated results during this study have highlighted the importance to control such FE preparations for the potential contamination with their bacterial producer organism, including GMM

Additional file 4: of Development and validation of an integrated DNA walking strategy to detect GMO expressing cry genes

Alignement of the targeted Cry1Ab sequences from the T304–40 event sequenced from the PCR verification assay (1 and 2) with the reference sequence from the T304–40 event (reference) used for the design of the target-specific primers (surrounded by orange rectangles). (DOCX 506 kb

Additional file 6: of Development and validation of an integrated DNA walking strategy to detect GMO expressing cry genes

Sequences of oligonucleotides provided by the APAgeneTM GOLD Genome Walking Kit from BIO S&T. (DOCX 13 kb