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.
