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International audience; 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 ≤ 10 copies and LOQa ≤ 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

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.

T25 is one of the 4 maize transformation events from which commercial lines have so far been authorized in Europe. It was created by polyethylene glycol-mediated transformation using a construct bearing one copy of the synthetic pat gene associated with both promoter and terminator of the 35S ribosomal gene from cauliflower mosaic virus. In this article, we report the sequencing of the whole T25 insert and the characterization of its integration site by using a genome walking strategy. Our results confirmed that one intact copy of the initial construct had been integrated in the plant genome. They also revealed, at the 5′ junction of the insert, the presence of a second truncated 35S promoter, probably resulting from rearrangements which may have occurred before or during integration of the plasmid DNA. The analysis of the junction fragments showed that the integration site of the insert presented high homologies with the Huck retrotransposon family. By using one primer annealing in the maize genome and the other in the 5′ end of the integrat ed DNA, we developed a reliable event-specific detection system for T25 maize. To provide means to comply with the European regulation, a real-time PCR test was designed for specific quantitation of T25 event by using Taqman® chemistry.

A highly sensitive quantitative real-time assay targeted on the 35S promoter of a commercial genetically modified organism (GMO) was characterized (sF/sR primers) and developed for an ABI Prism® 7700 Sequence Detection System and TaqMan® chemistry. The specificity assessment and performance criteria of sF/sR assay were compared to other P35S-targeted published assays. sF/sR primers amplified a 79 base pair DNA sequence located in a part of P35S that is highly conserved among many caulimovirus strains, i.e., this consensus part of CaMV P35S is likely to be present in many GM events. According to the experimental conditions, the absolute limit of detection for Bt176 corn was estimated between 0.2 and 2 copies of equivalent genome (CEG). The limit of quantification was reached below 0.1% Bt176 content. A Cauliflower Mosaic Virus control (CaMV) qualitative assay targeted on the ORF III of the viral genome was also used as a control (primers 3F/3R) to assess the presence of CaMV in plant-derived products. The specificity of this test was assessed on various CaMV strains, including the Figwort Mosaic Virus (FMV) and solanaceous CaMV strains. Considering the performance of sF/sR quantification test, the highly conserved sequence, and the small size of the amplicon, this assay was tested in a collaborative study in order to be proposed as an international standard.

Poster *INRA-PMDV route de Saint-Cyr 78026 Versailles-Grignon Diffusion du document : INRA-PMDV route de Saint-Cyr 78026 Versailles-Grignon; International audience

International audience