Report on the In-house Validation of a DNA Extraction Method from Soybean Grains and Validated Method

In accordance with relevant EU legislation , Dow AgroSciences LLC provided to the European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF) a DNA extraction method for soybean grains and the relevant samples (ground soybean grains). In line with its mandate , the EU RL GMFF has conducted an in-house validation of this DNA extraction method. To this end it tested the DNA extraction method on the samples provided and evaluated its performance in terms of DNA yield, integrity and quality. The in-house validation study confirmed that the method meets the method performance requirements as established by the EU-RL GMFF and the ENGL , and that it satisfies the provisions of Annex I-2.C.2 to Regulation (EC) No 641/2004. The method is therefore fit for the purpose of producing soybean DNA of suitable quantity and quality for subsequent PCR-based analysis.JRC.I.3-Molecular Biology and Genomic

Comparative Testing Report on the Detection and Quantification of GM events in compound feedstuff - Comparative testing round: ILC-EURL-GMFF-CT-02/12

The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF (Regulation (EC) No 1829/2003(1)) that is also mandated as EU-RL by Regulation (EC) 882/2006(2) organised a comparative testing round for National Reference Laboratories (NRLs) nominated under Regulation (EC) No 882/2004(2). Participation was open and free of charge for NRLs nominated under Regulation (EC) No 1981/2006(3), for all members of the European Network of GMO Laboratories (ENGL), and for official control laboratories from the EU and third countries. The EU-RL is accredited under ISO 17043 (‘General requirements for proficiency testing’(4)) and this comparative testing round met this ISO Standard(4). The test items were produced in-house from dried leaves of MON 88017 (MON-88Ø17-3) and seeds of soybean event 40-3-2 (MON-Ø4Ø32-6) provided by Monsanto, by spiking a compound feedstuff provided by a Belgian NRL. Participants were required to screen two test items (feedstuff Levels 1 and 2) for the presence of maize events Maize MON 88017, MON 89034 and soybean events 356043, 40-3-2 and MON 89788. Any event detected then had to be quantified. Participants could report the results in mass/mass % or copy/copy % and the EU-RL calculated the robust means (R) of Level 1 and 2 test items accordingly. In addition, "target" values () were assigned by the EU-RL on the basis of its homogeneity study(8) for m/m % data. These values were included in the uncertainty budget. The target standard deviation for CT was fixed by the Advisory Board for Comparative Testing at 0.15 (log10 value) for soybean event 40-3-2 and at 0.20 for maize event MON 88017 based on experience from previous CT rounds. This target standard deviation was used to derive z-scores for the participants’ results. Ninety laboratories from 43 countries registered for this CT round of which 82 from 35 countries returned at least qualitative test results. The results of the qualitative evaluation of the GM content indicated that most of the laboratories correctly detected soybean event 40-3-2 and maize event MON 88017 thus resulting in a very good performance overall. The results of the quantitative evaluation of GM content were found to be satisfactory overall for both events, with 94% of the laboratories submitting results in mass/mass % with a z-score, estimated on the basis of the robust mean, lying within the range of -2 to +2. This percentage decreased to 79% for results expressed in copy/copy %. When asked to repeat experimental work, most of the underperforming laboratories obtained satisfactory results. Only ~57% of participants provided information on measurement uncertainty in a complete and consistent manner, it is apparent therefore that despite the overall satisfactory outcome of this CT round, there is still improvement needed in this crucial area.JRC.I.3-Molecular Biology and Genomic

The analysis of food samples for the presence of Genetically Modified Organisms - User Manual

The User Manual - background information and didactical guide for the participants attending the training courses on ‘The analysis of Food Samples for the Presence of Genetically Modified Organisms’ organised by the Joint Reseach Centre - provides the theoretical and detaied practical information on the methodologies and protocols for GMO detection used during the training. Structured in 12 Sessions, it covers a wide variety of techniques for the detection, identification, characterisation, and quantification of GMO.JRC.F.7-Knowledge for Health and Consumer Safet

Comparative Testing Report on the Detection and Quantification of GM Events in Rice Noodles

The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF), accredited under ISO/IEC 17043, organised a comparative testing (CT) round for National Reference Laboratories (NRLs) nominated under Regulation (EC) No 882/2004, with voluntary participation of other official control laboratories. The test items consisted of rice noodles and commercial soybeans spiked with ground powder of soybean event DP-356043-5 in two different concentrations (Level 1 and 2). Participants were required to perform species identification and test for the presence of any GM event in the two test items. Any event detected then had to be quantified. Participants could report the results in mass/mass % or copy/copy % and the EU-RL GMFF calculated the robust means (R) for Level 1 and 2 test items accordingly. The target standard deviation for CT was fixed by the Advisory Board for Comparative Testing at 0.2 for the event, based on the experience of previous CT rounds. The robust means and target standard deviation were used to derive z-scores for the participants’ results. Eighty-eight laboratories from 42 countries registered for this CT round, of which 85 laboratories from 41 countries returned at least qualitative test results. When performing species identification, almost all laboratories correctly identified soybean and rice in both test items, and a few laboratories also detected maize and/or oilseed rape. In total 71 laboratories reported the presence of GM material in the test items, but 14 laboratories failed in this task. All of the 71 laboratories, except eight, correctly identified soybean event DP-356043-5 in the test items. Results of the quantitative evaluation of the GM content were satisfactory for both measurement units, with only two NRLs appointed under Regulation (EC) No 1981/2006 (one measuring in m/m % and one in cp/cp %) obtaining unsatisfactory z-scores (|z| ≥ 2.0) for both test items. Despite the overall satisfactory outcome of this CT round, only 58 % of participants provided information on measurement uncertainty in a complete and consistent manner, and further improvement in this crucial area is needed.JRC.I.3-Molecular Biology and Genomic

Report on the Verification of the Performance of Bt11, DAS-59122-7, MIR604, TC 1507 and GA21 Event-specific PCR-based Methods Applied to DNA Extracted from GM Stack Bt11 x DAS-59122-7 x MIR604 x TC 1507 x GA21 Maize

An application was submitted by Syngenta Crop Protection AG to request the authorisation of genetically modified stack (GM stack) Bt11 x 59122 x MIR604 x TC1507 x GA21 maize (tolerant to herbicide products containing glufosinate ammonium/glyphosate and resistant to certain lepidopteran/coleopteran pests) and all sub-combinations of the individual events as present in the segregating progeny (except for 1507 x 59122), for food and feed uses, and import and processing, in accordance with articles 5 and 17 of Regulation (EC) No 1829/2003 on GM Food and Feed. The unique identifier assigned to GM stack Bt11 x 59122 x MIR604 x TC1507 x GA21 maize is SYN-BTØ11-1 × DAS-59122-7 × SYN-IR6Ø4-5 × DAS-Ø15Ø7-1 × MON-ØØØ21-9. The GM stack Bt11 x 59122 x MIR604 x TC1507 x GA21 maize has been obtained by conventional crossing of genetically modified maize events: Bt11, 59122, MIR604, TC1507 and GA21, without any new genetic modification. The European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF) has previously validated individually, and declared fit for purpose, the detection methods for the single events Bt11, 59122, MIR604, TC1507 and GA21 (see http://gmo-crl.jrc.ec.europa.eu/StatusOfDossiers.aspx). In line with the approach defined by the European Network of GMO Laboratories (ENGL) (http://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Analytical_methods.pdf) the EU-RL GMFF has carried out only an in-house verification of the performance of each validated method when applied to genomic DNA extracted from GM stack Bt11 x 59122 x MIR604 x TC1507 x GA21 maize. The results of the in-house verification led to the conclusion that the individual methods meet the ENGL performance criteria also when applied to genomic DNA extracted from the GM stack Bt11 x 59122 x MIR604 x TC1507 x GA21 maize.JRC.I.3-Molecular Biology and Genomic

Comparative Testing Report on the Detection and Quantification of GM events in biscuit powder; Comparative testing round: ILC-EURL-GMFF-CT-01/13 - Version b

The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF), accredited under ISO 17043, organised a comparative testing (CT) round for National Reference Laboratories (NRLs) nominated under Regulation (EC) No 882/2004, with voluntary participation of other official control laboratories. The test items consisted of biscuit powder made from conventional maize and wheat grain spiked with powder of 98140 maize and MON 863 X MON 810 maize in different concentrations (Level 1 and 2). Participants were required to perform species identification and test for the presence of maize events 3272, Bt11, Bt176, 59122, GA21, MIR604, MON 810, MON 863, NK603, 1507, MON 88017, MON 89034, 98140 and MIR162 in the two test items. Any event detected then had to be quantified. Results could be reported in mass/mass % or copy/copy % and the EU-RL calculated the robust means (R) of Level 1 and 2 test items accordingly. The target standard deviation for CT was fixed by the Advisory Board for Comparative Testing at 0.20 for all events, based on the experience of previous CT rounds. The robust means and target standard deviation were used to derive z-scores for the participants’ results. Sixty-four laboratories from 28 countries registered for this CT round, of which all but one returned at least qualitative test results. The EU-RL GMFF issued a final report on this CT round in April 2014. After the final report was prepared, it was discovered that the distribution of the quantitative data obtained from laboratories for event MON 863 deviated from normality because some laboratories used the adh1 reference gene (with target amplicon of 70 bp) for quantification of MON 863 maize, which is known to carry a nucleotide polymorphism that may affect amplification. Therefore, the robust means for event MON 863 were re-calculated based on the data from laboratories that had used a reference gene different from adh1-70 bp. The original final report has been re-called; the current version contains the re-calculated z-scores for maize event MON 863. When performing species identification, almost all laboratories correctly identified maize species in each test item. In addition, approximately half of laboratories also identified soybean and a few oilseed rape. The majority of laboratories correctly detected the three GM events included in the Level 1 and Level 2 test samples. Results of the quantitative evaluation of the GM content were compromised by the use of the adh1 gene for MON 863 quantification by a number of laboratories. Forty five laboratories (71 %) obtained satisfactory z-scores (|z| ≤ 2.0) for the three GM events in both test items. Among the remaining 18 laboratories, half had unsatisfactory z-scores for MON 863. Only 46 % of participants provided information on measurement uncertainty in a complete and consistent manner and further improvement in this crucial area is needed.JRC.I.3-Molecular Biology and Genomic

Comparative Testing Report on the Detection and Quantification of Soybean 40-3-2 and Maize MON 88017 Comparative testing round: ILC-EURL-GMFF-CT-02/12 - Preliminary report

In the frame of Regulation (EC) No 882/2004, the European Union Reference Laboratory for Genetically Modified Food and Feed has the duty to organise comparative testing rounds and to ensure an appropriate follow-up of these activities. This preliminary report describes the outcome of the sixth comparative testing round ILC-EURL-GMFF-CT-02/12. Participants were required to screen two test items denoted compound feedstuff levels 1 and 2, for the presence of maize events Maize MON 88017, MON 89034 and soybean events 356043, 40-3-2 and MON 89788. Any events detected were then to be quantified. The test items were produced in-house. The EU-RL GMFF managed the on-line registration and submission of results. A total of 160 laboratories were invited to participate in ILC-EURL-GMFF-CT-02/12. Participants could report the results of the exercise either in mass/mass % or in copy/copy %. Overall, in this sixth comparative testing round results expressed in m/m % were characterised by a higher percentage of z-scores lying within the working range of -2 to +2 with respect to results reported in cp/cp % (mean 94.37 % versus 78.76 %, estimated only on z-scores calculated on the basis of the robust means). However, The differences in the percentages of z-scores within the working range between m/m % and cp/cp % results for maize event MON88017 (mean 96.08 % versus 71.57 % for m/m % and cp/cp % respectively, estimated only on Z calculated on the basis of the robust means) appear more relevant than what can be seen for soybean event 40-3-2 (mean 92.66 % versus 85.95 % for m/m % and cp/cp % respectively, estimated only on z-scores calculated on the basis of the robust means). Moreover, a relevant difference between the assigned value and the robust mean for level 2 concentration of maize event MON 88017 was observed. Such a difference occurred because of a substantial under-estimation of the actual concentration level by 43 out of 51 laboratories (i.e. 84.31 %) reporting results in m/m % format.JRC.I.3-Molecular Biology and Genomic

Metabolomic Profile at Birth, Bronchiolitis and Recurrent Wheezing: A 3-Year Prospective Study

There is growing interest for studying how early-life influences the development of respiratory diseases. Our aim was to apply metabolomic analysis to urine collected at birth, to evaluate whether there is any early metabolic signatures capable to distinguish children who will develop acute bronchiolitis and/or recurrent wheezing. Urine was collected at birth in healthy term newborns. Children were followed up to the age of 3 years and evaluated for the development of acute bronchiolitis and recurrent wheezing (≥3 episodes). Urine were analyzed through a liquid-chromatography mass-spectrometry based untargeted approach. Metabolomic data were investigated applying univariate and multivariate techniques. 205 children were included: 35 had bronchiolitis, 11 of whom had recurrent wheezing. Moreover, 13 children had recurrent wheezing not preceded by bronchiolitis. Multivariate data analysis didn’t lead to reliable classification models capable to distinguish children with and without bronchiolitis or with recurrent wheezing preceded by bronchiolitis neither by PLS for classification (PLS2C) nor by Random Forest (RF). However, a reliable signature was discovered to distinguish children who later develop recurrent wheezing not preceded by bronchiolitis, from those who do not (MCCoob = 0.45 for PLS2C and MCCoob = 0.48 for RF). In this unselected birth cohort, a well-established untargeted metabolomic approach found no biochemical-metabolic dysregulation at birth associated with the subsequent development of acute bronchiolitis or recurrent wheezing post-bronchiolitis, not supporting the hypothesis of an underlying predisposing background. On the other hand, a metabolic signature was discovered that characterizes children who develop wheezing not preceded by bronchiolitis

Airway metabolic anomalies in Adolescents with Bronchopulmonary Dysplasia: new insights from the metabolomic approach

Objectives To assess a group of adolescents with bronchopulmonary dysplasia (BPD) from a biochemicalmetabolic standpoint, applying the metabolomic approach to studying their exhaled breath condensate (EBC). Study design Twenty adolescents with BPD (mean age 14.8 years) and 15 healthy controls (mean age 15.2 years) were recruited for EBC collection, exhaled nitric oxide measurement, and spirometry. The EBC samples were analyzed using a metabolomic approach based on mass spectrometry. The obtained spectra were analyzed using multivariate statistical analysis tools. Results A reliable Orthogonal Projections to Latent Structures-Discriminant Analysis model showed a clear discrimination between cases of BPD and healthy controls (R2 = 0.95 and Q2 = 0.92). The search for putative biomarkers identified an altered complex lipid profile in the adolescents with BPD. Conclusions The metabolomic analysis of EBC distinguishes cases of BPD from healthy individuals, suggesting that the lung of survivors of BPD is characterized by long-term metabolic abnormalities. The search for putative biomarkers indicated a possible role of an altered surfactant composition, which may persist far beyond infancy.JRC.F.4-Fraud Detection and Preventio