Patrick Guertler1, Lutz Grohmann2, Heike Naumann3, Melanie Pavlovic1, Ulrich Busch1
1) Bavarian Health and Food Safety Authority, Germany;
2) Federal Office of Consumer Protection and Food Safety, Germany;
3) Lower Saxony State Office for Consumer Protection and Food Safety, Germany;
Genetically modified (gm) plants (GMP) have gained importance since commercialization in 1996. Cultivation areas increased from 1.7 million hectares in 1996 to almost 190 million hectares in 2017. In Europe, GMPs need to be authorized before being placed on the market and food and feed products containing authorized GMPs need to be labeled above a gm content of 0.9 %. Non-authorized products must not be placed on the EU market. One of the emerging GMP species is alfalfa (Medicago sativa), which is one of the most important forage crops worldwide. Modified gm alfalfa events J101 and J163 gained herbicide tolerance against glyphosate by incorporating a CTP2-CP4 epsps gene. In event KK179, the RNA interference technique was used to knock-out the caffeoyl-CoA-3-O-methyltransferase(CCOMT) translation. CCOMT is a key enzyme in the lignin pathway and a knock-out leads to an improved digestibility for ruminants. Gm alfalfa is commercially cultivated in the US and in Canada. In order to develop a qPCR-based detection method, we designed plasmids for each gm alfalfa event, based on published patent sequences. Further, we designed primers and a hydrolysis probe targeting the junction sequence spanning the plant genome and the transgenic insert (=event-specific detection). Plasmids were quantified using ddPCR and used for optimization and in-house validation of the methods. An estimated LOD95% of 10 copies per PCR was observed and PCR efficiencies of 95 – 97 % were achieved. Different qPCR instruments and PCR conditions were applied to test for robustness. Certified reference material for different GMP was used to test for specificity. No unspecific amplification signal was observed for any of the developed methods. An inter-laboratory comparison study with seven participating laboratories was conducted to show the transferability and applicability of the methods and to verify the assay performance parameters. Our cooperation partner (Federal Office of Consumer Protection and Food Safety, Berlin) was able to procure ground seed material for all three gm alfalfa events, which could be used in this inter-laboratory comparison study. All participants reported qPCR efficiencies between 95.9 % and 106.9 % and all laboratories were able to detect 10 nominal copies in twelve replicates. All results underline the suitability of the methods for the specific detection of gm alfalfa events J101, J163 and KK179.
A full collaborative trial validation study of the developed methods is planned for 2019.
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