Maddalena Querci

New approaches in GMO detection.

The steady rate of development and diffusion of genetically modified plants and their increasing diversification of characteristics, genes and genetic control elements poses a challenge in analysis of genetically modified organisms (GMOs). It is expected that in the near future the picture will be even more complex. Traditional approaches, mostly based on the sequential detection of one target at a time, or on a limited multiplexing, allowing only a few targets to be analysed at once, no longer meet the testing requirements. Along with new analytical technologies, new approaches for the detection of GMOs authorized for commercial purposes in various countries have been developed that rely on (1) a smart and accurate strategy for target selection, (2) the use of high-throughput systems or platforms for the detection of multiple targets and (3) algorithms that allow the conversion of analytical results into an indication of the presence of individual GMOs potentially present in an unknown sample. This paper reviews the latest progress made in GMO analysis, taking examples from the most recently developed strategies and tools, and addresses some of the critical aspects related to these approaches.

Applicability of the “Real-Time PCR-Based Ready-to-Use Multi-Target Analytical System for GMO Detection” in processed maize matrices

Simple tools to detect the presence of genetically modified organisms (GMO) in commercial products represent a valuable aid in managing the legal requirements for GMO testing in a cost-effective way. The “Real-Time PCR-Based Ready-to-Use Multi-Target Analytical System for GMO Detection” was developed to meet such requirements and was here further tested for its applicability on detecting GMO in recalcitrant maize matrices. Sixty-four processed maize products were purchased from the market of the European Union and analyzed for their GMO content. Seventy-five percent of the test samples were positive for the presence of GMO. In two samples, trace amounts of the so-called asynchronously authorized GMO could be detected. The overall outcome of the analyses indicated that most of the commercially available genetically modified maize events for food use could be detected in this study. Finally, the possibility to use the “Real-Time PCR-Based Ready-to-Use Multi-Target Analytical System for GMO Detection” in detecting GMO at a 0.1% mass level is documented. The implications of these results on the further development of such type of PCR-based GMO detection systems are discussed.

Development of an innovative immunoassay for CP4EPSPS and Cry1AB genetically modified protein detection and quantification.

An innovative immunoassay, called enzyme-linked immunoabsorbant assay (ELISA) Reverse, based on a new conformation of the solid phase, was developed. The solid support was expressly designed to be immersed directly in liquid samples to detect the presence of protein targets. Its application is proposed in those cases where a large number of samples have to be screened simultaneously or when the simultaneous detection of different proteins is required. As a first application, a quantitative immunoassay for Cry1AB protein in genetically modified maize was optimized. The method was tested using genetically modified organism concentrations from 0.1 to 2.0%. The limit of detection and limit of quantitation of the method were determined as 0.0056 and 0.0168 (expressed as the percentage of genetically modified organisms content), respectively. A qualitative multiplex assay to assess the presence of two genetically modified proteins simultaneously was also established for the case of the Cry1AB and the CP4EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) present in genetically modified maize and soy, respectively.

Novel nuclear barcode regions for the identification of flatfish species

The development of an efficient seafood traceability framework is crucial for the management of sustainable fisheries and the monitoring of potential substitution fraud across the food chain. Recent studies have shown the potential of DNA barcoding methods in this framework, with most of the efforts focusing on using mitochondrial targets such as the cytochrome oxidase 1 and cytochrome b genes. In this article, we show the identification of novel targets in the nuclear genome, and their associated primers, to be used for the efficient identification of flatfishes of the Pleuronectidae family. In addition, different in silico methods are described to generate a dataset of barcode reference sequences from the ever-growing wealth of publicly available sequence information, replacing, where possible, labour-intensive laboratory work. The short amplicon lengths render the analysis of these new barcode target regions ideally suited to next-generation sequencing techniques, allowing characterisation of multiple fish species in mixed and processed samples. Their location in the nucleus also improves currently used methods by allowing the identification of hybrid individuals.

A Ready-To-Use Multi-Target Analytical System for GM Soy and Maize Detection for Enforcement Laboratories

Today GMO analysis has become an integral part in the development of new genetically modified plants, in subsequent breeding, seed production and verification programmes. GMO analysis is a key technology in export and import of agricultural commodity products, for ascertaining regulatory compliance of GMOs in different countries, for labelling requirements, and for product authenticity and traceability. In Europe, in particular, GMO analysis is implemented in all Member States to fulfil legal requirements regarding GMOs and GMO-derived products, their release into the environment, cultivation, importation and their utilisation as food, food ingredients and animal feed (European Commission 2001, 2003a, 2003b). A key technical element for the authorisation of GMOs within the EU is the provision of an event-specific quantitative detection method validated according to internationally accepted standards to allow the control and monitoring of a particular GMO along the production, processing and the distribution chain. In this context the European Commission Joint Research Centre (JRC) has the mission to provide scientific and technical support to EU policy development on GMO. The nominated European Union Reference Laboratory for GM Food and Feed (EURL-GMFF) at the JRC is working at the forefront of GMO analysis in Europe. Legally responsible for method validation under Regulation (EC) No 1829/2003 (European Commission 2003a), the EURLGMFF works in close collaboration with the European Network of GMO Laboratories not only in method validation but also in the harmonisation of a number of topics spanning from sampling to method development, data analysis and measurement of uncertainty. In addition, in Europe, the EURL-GMFF plays a key role in the official food controls according to Regulation (EC) No 882/2004 (European Commission 2004a). Still, the multi-factorial nature of GMO analysis, the diverse and often complex composition of the samples under examination, the increasing number of GM events and, finally, the