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

Fraiture Marie-Alice, Guiderdoni Emmanuel, Meunier Anne Cecile, Papazova Nina, Roosens Nancy. 2022. ddPCR strategy to detect a gene-edited plant carrying a single variation point: Technical feasibility and interpretation issues. Food Control, 137:108904, 10 p.

Résumé : Gene-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.

Mots-clés Agrovoc : Oryza sativa, technique analytique, génie génétique, PCR, séquence nucléotidique, organisme génétiquement modifié, Gossypium hirsutum, distribution géographique

Mots-clés libres : Crispr/Cas9, Single nucleotide mutation, Transgenic rice, Detection, Digital droplet PCR, Food and feed chain

Champ stratégique Cirad : CTS 3 (2019-) - Systèmes alimentaires

Agences de financement hors UE : Belgian Federal Public Service of Health, Food Chain Safety, Agence Nationale de la Recherche

Projets sur financement : (BEL) Development and evaluation of approaches for detection of organisms modified by new genome editing techniques, (FRA) Ingénierie cellulaire : amélioration et innovation technologiques pour les plantes d'une agriculture

Auteurs et affiliations

• Fraiture Marie-Alice, Sciensano (BEL)
• Guiderdoni Emmanuel, CIRAD-BIOS-UMR AGAP (FRA)
• Meunier Anne Cecile, CIRAD-BIOS-UMR AGAP (FRA)
• Papazova Nina, Sciensano (BEL)
• Roosens Nancy, Sciensano (BEL) - auteur correspondant

Source : Cirad-Agritrop (https://agritrop.cirad.fr/603997/)

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