Maher Chaouachi

Abstract: The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic section of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and procumbentes) suitable for quantification of genetically modified (GM) sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R2> 0.994) over sugar beet standard concentrations ranging from 20000 to 10 HGC of sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory (EU-RL). This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.

Abstract: Reliable quantitative methods are needed to comply with current EU regulations on the mandatory labeling of genetically modified organisms (GMOs) and GMO-derived food and feed products with a minimum GMO content of 0.9 %. The implementation of EU Commission Recommendation 2004/787/EC on technical guidance for sampling and detection which meant as a helpful tool for the practical implementation of EC Regulation 1830/2003, which states that "the results of quantitative analysis should be expressed as the number of target DNA sequences per target taxon specific sequences calculated in terms of haploid genomes". This has led to an intense debate on the type of calibrator best suitable for GMO quantification. The main question addressed in this review is whether reference materials and calibrators should be matrix based or whether pure DNA analytes should be used for relative quantification in GMO analysis. The state of the art, including the advantages and drawbacks, of using DNA plasmid (compared to genomic DNA reference materials) as calibrators, is widely described. In addition, the influence of the genetic structure of seeds on real-time PCR quantitative results obtained for seed lots is discussed. The specific composition of a seed kernel, the mode of inheritance, and the ploidy level ensure that there is discordance between a GMO % expressed as a haploid genome equivalent and a GMO % based on numbers of seeds. This means that a threshold fixed as a percentage of seeds cannot be used as such for RT-PCR. All critical points that affect the expression of the GMO content in seeds are discussed in this paper.

Abstract: Plus de 30 espèces de poissons ont fait l’objet de travaux de transformation génétique depuis 1984, date de la production du premier poisson rouge transgénique (Carassius auratus auratus L. 1758). Bien que ces améliorations présentent des avantages dans le secteur de l’aquaculture et de l’environnement, aucun poisson transgénique n’a encore été commercialisé à des fins alimentaires dans le monde contrairement aux plantes génétiquement modifiées (GM). Cette possibilité de commercialisation est en cours d’étude par la FDA (Food and Drug Administration) concernant le saumon GM AquaAdvantage® de la firme AquaBounty.

Abstract: Abstract Since 2001, the traceability and labelling of genetically modified organism (GMO) food and feed derived products are obligatory in the European Union. Genetically modified organisms (GMO) are commonly detected via PCR tests. These tests typically involve several steps: (1) screening (2) construct specific (3) event specific and (4) reference gene. Screening tests are based on sequences frequently used for GM development,

Abstract: A high-throughput multiplex assay for the detection of genetically modified organisms (GMO) was developed on the basis of the existing SNPlex method designed for SNP genotyping. This SNPlex assay allows the simultaneous detection of up to 48 short DNA sequences ( approximately 70 bp; "signature sequences") from taxa endogenous reference genes, from GMO constructions, screening targets, construct-specific, and event-specific targets, and finally from donor organisms. This assay avoids certain shortcomings of multiplex PCR-based methods already in widespread use for GMO detection. The assay demonstrated high specificity and sensitivity. The results suggest that this assay is reliable, flexible, and cost- and time-effective for high-throughput GMO detection.

Abstract: The labeling of products containing Genetically Modified Organisms (GMO) is linked to their quantification since a threshold for the presence of fortuitous GMOs in food has been established. This threshold is calculated from a combination of two absolute quantification values: one for the specific GMO target and the second for an endogenous reference gene specific to the taxon. Thus, the development of reliable methods to quantify GMOs using endogenous reference genes in complex matrixes such as food and feed is needed. Plant identification can be difficult in the case of closely related taxa, which moreover are subject to introgression events. Based on the homology of β-fructosidase sequences obtained from public databases, two couples of consensus primers were designed for the detection, quantification and differentiation of four Solanaceae: potato (Solanum tuberosum), tomato (Solanum lycopersicum), pepper (Capsicum annuum) and eggplant (Solanum melongena). Sequence variability was studied first using lines and cultivars (intra-species sequence variability), then using taxa involved in gene introgressions and finally, using taxonomically close taxa (inter-species sequence variability). This study allowed us to design four highly specific TaqMan-MGB probes. A duplex real time PCR assay was developed for simultaneous quantification of tomato and potato. For eggplant and pepper, only simplex real time PCR tests were developed. The results demonstrated the high specificity and sensitivity of the assays. We therefore conclude that β-fructosidase can be used as an endogenous reference gene for GMO analysis.

Abstract: Due to its very large use in the first generation of genetically modified organisms (GMO), the 35S promoter derived from the cauliflower mosaic virus (CaMV) is the most used PCR target for screening tests in GMO routine analysis, before any identification and quantification of GMOs. Accordingly, a specific detection of the virus donor organism is required to avoid false positives. A new qualitative and quantitative method based on real time polymerase chain reaction (PCR) techniques was developed for the detection and quantification of CaMV. The region targeted was an internal part of a qualitative test previously published using the ORFIII of the CaMV genome. It codes for a protein (P3) responsible for the pathogen infectivity and not in the ORFIV coding for a coat protein (P4), which can be used for GMO constructions. In this paper, we show the high reliability of the PCR test both in simplex and duplex (with P35S of the CaMV). This test shows high specificity and sensitivity. (LODa B 10 copies and LOQa B 100 copies). Advantages and drawbacks of this test with a previously published test are discussed. Finally, the ORFIII PCR product was cloned in a pGEM-T vector for further use in the near future as an alternative calibrant for quantitative analyses through its availability from the international BCCM/ LMBP Ghent plasmid bank.

Abstract: In the first part of the paper, we report the description of a new strategy for the development of a plant reference gene system that can be used for genetically modified organism (GMO) analysis. On the basis of in silico research for candidate genes, the design of degenerate primers allowed the obtention of genomic sequences of the selected gene ppi-phosphofructokinase ( ppi-PPF) for nine taxa in which GMOs have been developed. The comparison and the analysis of inter- and intraspecies sequence variability were performed using a large number of species and cultivars. As an example of application following the detection of single nucleotide polymorphism, we designed specific conventional and real-time polymerase chain reaction tests for the detection and quantification of three taxa, namely, maize, cotton, and rice. This system was highly specific and sensitive. The gene copy number conservation among different cultivars was analyzed and confirmed with a sequencing step. This reference gene system is adequate for use in routine assays for the quantification of GMOs. We then explain briefly the constraints faced and propose recommendations when designing a reference gene system depending on the species to be targeted.

Abstract: Face à la croissance des surfaces cultivées en OGM dans le monde, comment savoir si les produits que nous consommons sont dérivés de plantes génétiquement modifiées ? Quels sont les outils disponibles aujourd’hui qui permettent de le savoir ? En Europe, plusieurs laboratoires et programmes de recherche ont tenté ces dernières années de répondre à ces questions.

Abstract: De nombreux pays ont mis en place une réglementation pour contrôler et détecter la présence du matériel génétiquement modifié (GM) dans des différentes matrices (semences, grains et produits dérivés). En Europe, un seuil de 0.9% a été fixé pour étiqueter les produits GM. Ceci a nécessité le développement de méthodes pour la détection et la quantification des OGM en se basant sur deux cibles moléculaires principales, la protéine ou l’ADN. Actuellement, les séquences des deux sites d’intégrations 3’ et 5’ du maïs transgénique Bt11 ont été décrites. On a utilisé ces informations pour la définition d’un nouveau test spécifique pour la détection et la quantification de cet OGM en se basant sur la PCR qualitative et quantitative en temps réel (RT-PCR, Chimie TaqMan). La quantification relative de l’événement Bt11 a été effectuée en se référant à un gène endogène du maïs, l’alcool deshydrogenase (adh1). Les résultats ont été exprimés en pourcentages des standards connus du maïs Bt11 rapportés à la quantité totale de maïs. Beaucoup de paramètres de performances ont été vérifiés. Le test développé s’est avéré très spécifique et d’une grande sensibilité.