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

An innovative integrated approach based on DNA walking to identify unauthorized GMOs

&lt;p&gt;In the next coming years, the frequency of unauthorized genetically modified organisms (GMOs) being present in the European food/feed chain will increase significantly. Rice already constitutes a challenge for laboratories developing methods to detect unauthorized GMOs. Indeed, in 2012, several genetic modified rices were detected in products imported from Asia, mainly from China. Therefore, we have developed a strategy to identify unauthorized GMOs containing a pCAMBIA family vector, frequently present in transgenic plants. The presented integrated approach is performed in two main successive steps on Bt rice grains. First, the potential presence of unauthorized GMOs is assessed by the qPCR SYBR&amp;reg;Green technology targeting the terminator 35S (t35S) pCAMBIA element, which allows discriminating pCAMBIA family vectors. Second, its presence is confirmed via the characterization of the junction between the transgenic cassette and the rice genome. To this end, a DNA walking strategy is applied using a first reverse primer followed by two semi-nested PCR rounds using primers that are each time nested to the previous reverse primer. The sensitivity of the method was assessed. This innovative approach allows to rapidly identifying the transgene flanking region and presents the advantage to be easily implementable in GMO routine analysis by the enforcement laboratories.&lt;/p&gt;</p

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

UGM characterization using DNA walking strategy

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Development and validation of an integrated DNA walking strategy to detect GMO expressing cry genes

Abstract Background Recently, an integrated DNA walking strategy has been proposed to prove the presence of GMO via the characterisation of sequences of interest, including their transgene flanking regions and the unnatural associations of elements in their transgenic cassettes. To this end, the p35S, tNOS and t35S pCAMBIA elements have been selected as key targets, allowing the coverage of most of GMO, EU authorized or not. In the present study, a bidirectional DNA walking method anchored on the CryAb/c genes is proposed with the aim to cover additional GMO and additional sequences of interest. Results The performance of the proposed bidirectional DNA walking method anchored on the CryAb/c genes has been evaluated in a first time for its feasibility using several GM events possessing these CryAb/c genes. Afterwards, its sensitivity has been investigated through low concentrations of targets (as low as 20 HGE). In addition, to illustrate its applicability, the entire workflow has been tested on a sample mimicking food/feed matrices analysed in GMO routine analysis. Conclusion Given the successful assessment of its performance, the present bidirectional DNA walking method anchored on the CryAb/c genes can easily be implemented in GMO routine analysis by the enforcement laboratories and allows completing the entire DNA walking strategy in targeting an additional transgenic element frequently found in GMO

Development of a Taxon-Specific Real-Time PCR Method Targeting the <i>Bacillus subtilis</i> Group to Strengthen the Control of Genetically Modified Bacteria in Fermentation Products

Most of the bacteria that are used to produce fermentation products, such as enzymes, additives and flavorings, belong to the Bacillus subtilis group. Recently, unexpected contaminations with unauthorized genetically modified (GM) bacteria (viable cells and associated DNA) that were carrying antimicrobial resistance (AMR) genes was noticed in several microbial fermentation products that have been commercialized on the food and feed market. These contaminations consisted of GM Bacillus species belonging to the B. subtilis group. In order to screen for the potential presence of such contaminations, in this study we have developed a new real-time PCR method targeting the B. subtilis group, including B. subtilis, B. licheniformis, B. amyloliquefaciens and B. velezensis. The method’s performance was successfully assessed as specific and sensitive, complying with the Minimum Performance Requirements for Analytical Methods of GMO Testing that is used as a standard by the GMO enforcement laboratories. The method’s applicability was also tested on 25 commercial microbial fermentation products. In addition, this method was developed to be compatible with the PCR-based strategy that was recently developed for the detection of unauthorized GM bacteria. This taxon-specific method allows the strengthening of the set of screening markers that are targeting key sequences that are frequently found in GM bacteria (AMR genes and shuttle vector), reinforcing control over the food and feed chain in order to guarantee its safety and traceability

Development of a Taxon-Specific Real-Time Polymerase Chain Reaction Method to Detect <i>Trichoderma reesei</i> Contaminations in Fermentation Products

Recently, a genetically modified microorganism (GMM) detection strategy using real-time PCR technology was developed to control fermentation products commercialized in the food and feed chain, allowing several unexpected GMM contaminations to be highlighted. Currently, only bacterial strains are targeted by this strategy. Given that fungal strains, like Trichoderma reesei, are also frequently used by the food industry to produce fermentation products, a novel real-time PCR method specific to this fungal species was developed and validated in this study to reinforce the GMM detection strategy. Designed to cover a sequence of 130 bp from the translation elongation factor alpha 1 (Tef1) gene of T. reesei, this real-time PCR method, namely TR, allows for the screening of commercial fermentation products contaminated with T. reesei, genetically modified or not, which is one of the major fungal species used as an industrial platform for the manufacturing of fermentation products. The developed real-time PCR TR method was assessed as specific and sensitive (LOD95% = eight copies). In addition, the developed real-time PCR TR method performance was confirmed to be in line with the “Minimum Performance Requirements for Analytical Methods of GMO Testing” of the European Network of GMO Laboratories. The validated real-time PCR TR method was also demonstrated to be applicable to commercial microbial fermentation products. Based on all these results, the novel real-time PCR TR method was assessed as valuable for strengthening the current GMM detection strategy regarding major fungal species used by the food industry to produce microbial fermentation products