Analytes and Related PCR Primers Used for GMO Detection and Quantification

This document provides the first general overview on PCR primers used by Member State control laboratories for the detection, identification and quantification of GMOs. The data has been collected from a survey launched in 2005. The survey aimed at reviewing the PCR primers used by ENGL members for control purposes and at gathering analytical details on the corresponding PCR procedures. It further aimed to provide a catalogue of DNA sequences as a basis for the future development of plasmid standards. Participants to the survey were specifically requested to provide information on the genetic targets and if relevant, the GM event for which the primers were designed, to include the sequences of primers and the purpose (qualitative/quantitative and screening/identification analysis), type (i.e. single, multiplex, competitive, double-competitive, simplex real-time or multiplex real-time) and specificity (gene-specificity, construct specificity or event-specificity) of the corresponding PCR assay. They were further requested to provide comments and references, as well as to specify the confidentiality status of data provided. As a quality control step, the collated information was checked against the corresponding data published in the GMOs methods database (genetic elements were targeted in the PCR assays. The general overview revealed that 83% of the primers used by laboratories were published in peer-reviewed articles. Custom-designed primers or commercially available kits were rarely reported. The survey data further indicated that 44% of primers were used for detecting common transgenic elements and endogenous genes while the other primers were predominantly employed for identification of authorised GMO. The general overview also revealed that 46% of reported primers had been tested in collaborative studies. Further data analyses were performed on the sets of primers defined by the participating laboratories for a particular scope, purpose and specificity of the PCR assay. Strikingly, these analyses revealed a great variability of primers selection for GMOs control purposes. In general, however, a single primer pair was reported by more than 30% of the ENGL members while there was a wide distribution of primer utilisation in the remaining laboratories. The primer pairs most commonly employed were construct-specific, while for quantitative identification, ENGL laboratories tended to use primers that were event-specific. In approximately 30% of cases, primers designed for quantitative determination were also utilised for qualitative analysis. Primer pairs were designed predominantly for identification of GTS 40-3-2 and Event 176 which were the first GMOs approved for food in the EU. For new transgenic events most laboratories were using primers validated by the Community Reference Laboratory for GM Food and Feed (CRL-GMFF). This tendency is expected to continue in the future. For some GM events authorised in the EU (i.e. GT73, Falcon GS/40/90pHoe6/Ac, RF3, and MS8) no validated primers were available. Also, not all GM events that were authorised in the EU have had primers reported for corresponding detection/quantification. For identification of crops as ingredient the results indicated that a large number of taxon-specific primers were targeting maize genes. A second group of primers was specific for soybean with lectin as the only targeted gene. Taxon-specific primers were also reported for detection of canola, tomato, potato, wheat, cotton or chloroplast-specific sequences. In general, few of the reported taxon-specific primers have been subjected to ring-trial evaluation. This survey has provided a significant insight into the analytical strategies exploited by the ENGL laboratories for detection, identification and quantification of GMOs. It has shown that primer pairs used in the laboratories for the same final purpose are quite diverse, even if some of the primer pairs are used more frequently than others. Quality control of the data collected in the survey highlighted a problem of nomenclature for primers. Indeed, the same oligonucletide sequences had different formats and names in different ENGL laboratories. Use of the same nomenclature for primers should be promoted at ENGL level. In addition, the ENGL could drive harmonisation in the adoption of scientific and technical approaches for GMO analyses and provide recommendations for primers utilisation. The lack of performance criteria for the specificity of qualitative amplicons and methods exploiting them is certainly a weakness to address. Provision of reference amplicons and application of performance criteria to primers and probe utilisation should be conceived as an initial step in promoting coordination, standardisation and harmonisation in Member States. The mission of the JRC is to provide customer-driven scientific and technical support for the conception, development, implementation and monitoring of EU policies. As a service of the European Commission, the JRC functions as a reference centre of science and technology for the Union. Close to the policy-making process, it serves the common interest of the Member States, while being independent of special interests, whether private or national. LB -N A -2359-EN -C