Abstract: To overcome the difficulties of obtaining the Certified Reference Material (CRM) and according to the key documents of the European Union Reference Laboratory (EU-RL), a new standard reference molecule containing the construct specific of the canola event Oxy-235 (3′-junction Nitrilase/Tnos) and the canola endogenous reference gene (acety-CoA-carboxylase) was constructed and used for duplex real-time quantitative analysis. The limits of detection (LOD) were less than 5 Haploid Genome Copy (HGC) and the limits of quantification (LOQ) were about 10 HGC. Furthermore, mixed GM and non-GM canola samples were analysed with duplex QRT-PCR to evaluate the performance criteria as required for validation procedures in the EU-RL, namely, the precision and the accuracy. The accuracy expressed as bias ranged from 2% to 10% and the precision (repeatability and reproducibility) expressed as the RSDr and RSDR was from 2.2 to 5.12 and 2.15 to 5.46 respectively. All these indicated that the developed construct specific method and the reference molecule are suitable for the identification and the quantification of the canola event Oxy-235.
Abstract: Genetically modified organisms (GMO) invade more and more the agricultural production in the world. Although there are no legislations on GM labeling and cultivation of GM crops in Tunisia, the present study aims to check the status of GMO in Tunisian market using qualitative and quantitative real time-PCR (QRT-PCR). Three-hundred-sixty five samples were collected and different DNA extraction methods were adapted and optimized. Specific primers targeting 35S promoter from Cauliflower mosaic virus (CaMV) and nopaline synthase terminator from Agrobacterium tumefaciens (At) were used for the detection of the GMO insert and Taxon specific primers for the detection of plant species. Validated Taqman® probes (EU-RL) targeting event specific regions of the maize events MON810, Bt11, and the soybean event RRS were used for the quantification studies. Seven food and feed products showed different amounts of RRS (1.9%), MON810 (2.1%), and Bt11 (1.6%). The results demonstrate for the first time the presence of GMO in Tunisian markets reinforcing the need for the development of accurate quantitative methods in routine analyses.
Abstract: In response to the increasing number of genetically modified (GM) events released on the market, control laboratories explore various strategies to simplify and reduce the number of tests needed to characterise the content in genetically modified organism (GMO) of a given sample. Lastly, multiplexing is considered as one of the possible ways to decrease the time and cost of analysis. Here, we report the development of four duplex polymerase chain reaction (PCR) tests for the identification and the quantification of four maize transformation events from which commercial lines have been authorised in Europe namely, Bt11 and Bt176 (Syngenta, DE, USA), Mon810 MaisGard™ (Monsanto, MO, USA) and T25 Liberty Link™ (Bayer CropScience, Monheim, Germany). The duplex PCR tests combine a maize-specific PCR test hybridising in the Adh1 locus with an event-specific detection system designed on a junction fragment for each of these four GM maize. Realtime PCR tests, suitable to comply with the European regulation, were designed by using Taqman® chemistry.
PCR tests, suitable to comply with the European regulation,
were designed by using Taqman® chemistry.
Abstract: Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis. The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the 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 (R (2) > 0.994) over sugar beet standard concentrations ranging from 20,000 to 10 HGC of the 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. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.
Notes: Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis.
