Report on the Verification of the Performance of a Construct-Specific Assay for the Detection of Flax CDC Triffid Event FP967 Using Real-Time PCR

Further to the detection by the German authorities of the unauthorised flax CDC Triffid event FP967 (Unique Identifier CDC-FLØØ1-2) in materials imported from Canada, a notification was sent through the Rapid Alert System for Food and Feed (RASFF) in September 2009. On 21st August 2009, the Community Reference Laboratory for Genetically Modified Food and Feed received from the German authorities a construct-specific method for the detection of flax CDC Triffid event FP967, developed by Genetic ID, Augsburg (Germany). The method developer declared this method as specific for event FP967 as it targets a transition sequence spanning the nopaline synthase (nos) terminator gene and the spectinomycin/streptomycin resistance gene, construction being found only in the flax FP967 event. On 11th September 2009, the CRL-GMFF received from the German authorities the FP967 positive control in the form of DNA extracted from seeds. Seeds were provided to the German authorities by the University of California, Riverside, USA. The CRL-GMFF carried out experiments on the control sample received in order to verify the specificity and the Limit of Detection (LOD) of the construct-specific method. The CRL-GMFF observed that the NOST-Spec (nos terminator ¿ spectinomycin resistance gene) construct-specific method generates a PCR amplification product of 105 bp, whose sequence is homologous to a transition sequence spanning the nopaline synthase (nos) terminator gene and the dihydrofolate reductase gene. The experimental testing of the specificity indicates that the NOST-Spec construct-specific assay does not detect genetically modified events under the conditions reported. The limit of detection (LOD) established is between 1 and 5 haploid genome copies of FP967.JRC.DDG.I.4-Molecular biology and genomic

Cambiamenti ambientali indotti dalle variazioni climatiche oloceniche e dall’uomo nell’area dell’abitato antico di Pontecagnano

L’abitato antico di Pontecagnano (vii-iii a.C.), sorge su un alto morfologico di natura travertinosa, che in antico emergeva di pochi metri dal settore NO della pianura alluvionale costiera del Fiume Sele. I lavori per l’ampliamento dell’autostrada A3 SA-RC hanno intercettato livelli archeologici rappresentativi di ampie porzioni del territorio urbano e periurbano dell’abitato antico e messo in evidenza un record archeostratigrafico che va dal Pleistocene Superiore ad oggi. Lo studio geomorfologico ed archeo-tephro-stratigrafico di dettaglio, corredato da analisi paleoambientali, ha consentito di delineare gli aspetti salienti dell’evoluzione del paesaggio e degli ambienti nel corso dell’Olocene. Le modifiche dell’ambiente e del paesaggio sono state prevalentemente indotte da condizionamenti antropici sul sistema idraulico e forestale e sull’organizzazione del territorio soprattutto per il periodo di vita dell’abitato, dove si coglie un importante bonifica. Nei periodi precedenti e successivi alla vita dell’abitato i cambiamenti ambientali sono stati indotti da variazioni climatiche e dai prodotti delle eruzioni dei vulcani napoletani.The ancient settlement of Pontecagnano (7th-3rd centuries B.C.) was built up on the travertine plateau overlooking the Sele river on the NW sector of the alluvial-coastal plain. Motorway construction works brought to light archaeological remains of an ancient urban and suburban settlement. Archaeostratigraphical records dated between the late Pleistocene and today have been elucidated. The geomorphological and archaeo-tephro-stratigraphical study, coupled with palaeoenvironmental analysis, allowed us to outline the evolution of the environment during the Holocene. The environmental changes have been mainly induced by human activities, during the 7th -3rd centuries B.C., by land reclamation. During other periods of the Holocene the environmental changes can be attributed to climatic variations and, secondly, to the impact of the distal products of Neapolitan volcanic eruptions on geomorphic systems

Sviluppo ed Ottimizzazione di Metodi Analitici Innovativi Atti alla Quantificazione di Proteine Prodotte da Organismi Geneticamente Modificati (OGM) negli Alimenti

Secondo le norme vigenti (Food and Feed regulation 1829/2003/EC, aggiornamento No 641/2004 del 6 Aprile 2004) l'introduzione di un transgene nel mercato alimentare europeo può avvenire solo dopo l'invio di una specifica richiesta all'autorità competente e l'approvazione da parte della Commissione. Nel momento in cui si propone la commercializzazione di un nuovo OGM, un dossier contenente le informazioni richieste sulla natura del transgene, quali la descrizione, lo studio di impatto ambientale e sulla salute umana, le informazioni di natura molecolare e il metodo evento-specifico di identificazione e quantificazione dell'OGM devono essere incluse. E` importante sottolineare che attualmente è in vigore l'obbligo di etichettatura per i prodotti OGM. Le quantità massime di OGM definite in % di wOGM/wtotale che possono essere presenti nei cibi sono state così definite: per i trasgeni non autorizzati la tolleranza è zero, per quelli in via di autorizzazione e per soli tre anni è dello 0.5%, mentre per quelli autorizzati e` dello 0.9%. Ciò implica la possibilità da parte degli enti preposti al controllo di poter misurare correttamente il contenuto di OGM nei prodotti alimentari al fine di garantire il rispetto delle norme e la tracciabilità dei singoli ingredienti come richiesto dalla 1830/2003/EC. Attualmente in Europa i metodi evento specifici accettati e utilizzati sono tutti basati sulla Real Time PCR che permette di risalire ad un numero di copie di DNA target inserite nel genoma della specie in esame. Per rendere il metodo evento specifico un primer è complementare ad una sequenza del genoma della specie ospite del trasgene e l'altro primer è invece complementare ad una sequenza interna alla sequenza del gene inserito. Vengono così amplificate le zone di giunzione tra il genoma e il costrutto inserito. Malgrado il fatto che l'errore del saggio sia stimato intorno al 30% e il calcolo per ottenere un dato definitivo contiene diverse variabili, al momento rimane la tecnica di elezione in questo settore. I metodi immunologici non sono molto utilizzati in Europa perchè non sono saggi evento specifici ovvero non permettono di individuare la specie che porta il trasgene. Di contro sono molto più utilizzati nei paesi produttori di materie prime come il mais e la soia. Nei paesi produttori infatti i primi controlli (screening test) avvengono direttamente sul campo, in molti casi vengono utilizzati screening multipli soprattutto se in larga scala in quanto i costi sono molto contenuti rispetto alla RT-PCR (ISO 21572:2002). Un'altra problematica del sistema di monitoraggio degli OGM consiste nei metodi di campionamento attuabili, a tal proposito la Commissione europea ha emanato una Raccomandazione apposita (Recommendation EC No 787/2004). La possibilità di riconoscere e quantificare più OGM in un unico saggio è certamente di interesse anche in Europa considerando che nei prossimi cinque anni si attendono decine di nuovi OGM. La validazione di metodi multiplex, quantitativi, potenzialmente automatizzabili con costi contenuti sarebbe quindi auspicabile (ISO 21572:2002, ISO 5725:1994. IUPAC, 1997).JRC.I.6-Biotechnology and GMO

GMO Testing Methods: Analytical Approaches, Method Validation and Sampling Strategy

The detection and the identification of genetically modified organisms (GMOs) in seeds, grains and other materials are driven by legal requirements and marketing demands. In the EU ¿food and feed¿ Regulations [Reg. (EC) No 1829/2003, Reg. (EC) No 1830/2003] require compulsory labeling for all GMO-derived products including bulk commodities, seeds both for human consumption and animal feeding. The ¿food and feed¿ Regulation sets to 0.9% the threshold for the adventitious presence of authorized GMO-derived materials and 0% for non-authorized GMOs. In order to test the amount of GMOs in a consistent and reproducible way suitable analytical methods are required. Moreover, mandatory labeling is also required for those products where GMO-derived DNA or proteins are no longer detectable as for highly refined oils. In addition, traceability of GMOs has to be guaranteed in all products produced from GMOs at all stages of their placing on the market through the production and distribution chain¿ as required by Reg. (EC) No 1830/2003. In the process of GMO authorization an event-specific method for detection and identification of the transformation event must be provided by the applicants to the competent Authorities. In such respect, the Community Reference Laboratory for GM Food and Feed (CRL-GMFF) (established by Reg. (EC) No 1830/2003), has the mandate to validate the methods provided by the GMOs producers (http://gmo-crl.jrc.it/). All the CRL-GMFF validated methods are published in the GM Register (http://ec.europa.eu/food/food/biotechnology/authorisation/index_en.htm). Other methods for detection of GMOs are annexed to international standards (ISO 21569:2005, ISO 21570:2005, ISO 21572:2005). In this respect, and as a measure to implement legislation and facilitate stakeholders in quick retrieval of methods, a comprehensive database of methods has been established and maintained at Biotechnology and GMOs Unit of the European Commission¿s Institute for Health and Consumer Protection (IHCP), aimed at the diffusion of information on validated methods for detection and quantification of GMOs (http://biotech.jrc.it/methodsdatabase.htm).JRC.I.4-Molecular biology and genomic

Nickel, Cobalt and Chromium-Induced Cytotoxicity and Intracellular Accumulation in Human HaCaT Keratinocytes.

Abstract not availableJRC.(EI)-Environment Institut

Innovative Application of Fluorescent Micro-sphere Based Assay for Multiple GMO Detection

An innovative multiple screening protocol allowing the detection of specific DNA sequences of p35S and epsps in GM soya flours simultaneously was developed. For the application of the Luminex xMAP technology, two different sets of fluorescent beads were respectively cross-linked to the specific oligonucleotide probes previously amplified and labelled by PCR reaction in the presence of a biotinylated nucleotide. The detection of the amplification products bounded by the streptavidin-phycoeritrin conjugate was performed using the Luminex-100 instrument. The system allows to identify extremely low amounts of labelled targets as of 7.8 10-4 nmol of p35S antiprobe with an associated value of RSDr equal to 2.8. Parameters such as repeatability, reproducibility, RSD, LOD and LOQ were considered to evaluate the performances of the assay. The potentiality of the system here described, enables us to look forward to a multiple target assay able to simultaneously detect and eventually quantify tens of target sequences occurring within the same sample preparation. The present study describes the first application of a multi-target fluorescent micro-sphere based assay for DNA GMO detection.JRC.I.6-Biotechnology and GMO

A Qualitative Approach for the Assessment of the Genetic Stability of the MON 810 Trait in Commercial Seed Maize Varieties

Maize MON 810 is one of the European Union (EU) authorised genetically modified organisms (GMO) for placing on the food and feed market. The total number of MON 810 varieties registeredin the European Common Catalogues of varieties of agricultural plant species has almost tripled since 2005. One ofthe requirements described in EU legislation, namely the genetic stability of GM seed varieties, was thus assessed by analysing the intactness of the entire MON 810 integration and its genotypic stability in commercial varieties available on the market for at least the last two years. A combined strategy using qualitative analytical methods made possible to determine the presence/absence of the individual genetic elements and of the whole GM construct. The restriction fragment length polymorphism (RFLP)patterns obtained from amplified whole constructs by long PCR were compared side by side. CryIA(b) protein expression levels were determined by ELISA. Twenty-four out of the 26 analysed varieties met the expected stability features. One variety gave negative results in all assays and one variety contained the necessary genetic elements for expressing CryIA(b) protein, although giving negative results for the long PCR product. To our knowledge, this study is the first post-marketing stability analysis performed on GM commerical seed varieties.JRC.I.3-Molecular Biology and Genomic

Development of an innovative immunoassay for CP4EPSPS and Cry1AB GM proteins detection and quantification

An innovative immunoassay, called ELISA Reverse, based on a new conformation of the solid phase was developed; the solid support was expressly designed for being 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 GM maize was optimised. The method was tested using GMO concentrations from 0.1 to 2%. The LOD and LOQ of the method were respectively determined equal to 0.0056 and 0.0168 (expressed in % of GMO content). A protocol to assess the presence of two GM proteins simultaneously has also been established for the case of the Cry1AB and the CP4EPSPS respectively present in GM maize and soy.JRC.I.6-Biotechnology and GMO

Statistical Evaluation of Real-Time PCR Protocols Applied to Quantify Genetically Modified Maize

Real-Time PCR (RTi-PCR) is the technique of choice for event-specific quantification of GMOs by determining the amount of event with respect to a species-specific reference gene. Reference genes can be amplified from the genome extracted from Certified Reference Materials (CRMs) or from ad-hoc designed plasmids. In the present study, we statistically evaluate the performance of RTi-PCR protocols for GM maize MON810 event by using both genomic DNA from conventional CRMs and a plasmid containing sequences representative of four maize species-specific reference genes. The significance of simple and interaction effects of several variables included in the experimental design on DNA quantification methods and RTi-PCR were evaluated and discussed. Statistically significant differences on Ct values may have an impact on the GMOs quantification and consequently on the compliance of GM quantification established legal thresholds. Our results confirm the reliability of the plasmid as alternative calibrant for the calculation of GMOs copy number.JRC.I.3-Molecular Biology and Genomic