CERTIFICATION REPORT The certification of different mass fractions of VCO-Ø1981-5 in maize seed powder Certified Reference Materials ERM®-BF438a, ERM®-BF438b, ERM®-BF438c, ERM®-BF438d and ERM®-BF438e

This report describes the production of a set of Certified Reference Materials (CRMs) ERM BF438a, b, c, d and e, which are certified for their VCO-Ø1981-5 mass fractions. These materials were produced following ISO Guide 34:2009 and are certified in accordance with ISO Guide 35:2006. Genetically modified (GM) seeds of the maize event VCO-Ø1981-5 and seeds from a non-GM maize variety were milled to obtain GM and non-GM seed powders with a similar particle size distribution. Mixtures of non-GM and GM maize powder were prepared gravimetrically. The certified value was obtained from the gravimetric preparations, taking into account the genetic purity with respect to the event VCO-Ø1981-5 of the two powder materials and their water mass fractions. The certified values were confirmed by event-specific real-time PCR as an independent verification method (measurements were within the scope of accreditation to ISO/IEC 17025:2005). The uncertainties of the certified values were estimated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) [4] and include uncertainties relating to possible inhomogeneity (Section 4), instability (Section 5) and characterisation (Section 6). The materials are intended for the calibration or quality control of real-time PCR measurements to identify VCO-Ø1981-5 maize and/or quantify its mass fraction. As with any reference material, they can also be used for establishing control charts or for carrying out validation studies. The CRMs are available in glass bottles containing at least 1 g of dried maize seed powder, which were sealed under atmosphere of argon. The minimum amount of sample of the mixtures to be used and recommend for the blank and pure VCO-Ø1981-5 materials is 200 mg. The CRMs were accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium.JRC.D.2-Standards for Innovation and sustainable Developmen

Certification of Reference Materials of Soya Seed Powder with different Mass Fractions of genetically modified roundup ready® Soya, Certified Reference Materials ERM®-BF410k (ERM®-BF410ak, ERM®-BF410bk, ERM®-BF410dk, ERM®-BF410gk)

This report descirbes the processing and certification of four soya seed powder Certified Reference Materials (CRMs) containing different mass fractions of genetically modified (GM) GTS 40-30-2 (Roundup Ready) soya (ERM-BF410ak, bk, dk, gk), according to the principles of ISO Guide 34. The certification values were confirmed by event-specific real-time PCR as independent verification method. The CRMs are intended to be used for quality control or calibration of methods for the quantification of the GTS 40-30-2 soya mass fraction in food and feed. The four CRMs have been accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Material consortium.JRC.D.2-Reference material

CERTIFICATION REPORT: The certification of the mass of lambda DNA in a solution Certified Reference Material: ERM®-AD442k

This report describes the processing and certification of genomic lambda deoxyribonucleic acid (DNA) in a solution. ERM®-AD442k is certified for its lambda DNA mass, expressed in ng/µL. The DNA copy number concentration in cp/µL is provided as an indicative value. The material was produced according to ISO Guide 34:2009. A volume of approximately 400 mL of lambda DNA at an approximate concentration of 450 ng/µL was purchased from Promega Corporation and Benelux BV (Madison, USA and Leiden, NL). After homogenising and diluting this solution with TE buffer, 3100 vials of ERM-AD442k were produced. Each ERM-AD442k vial contains a certified DNA mass concentration of 57.53 ng/µL with an expanded combined uncertainty of 1.07 ng/µL. Using two different next generation sequencing (NGS) techniques (i.e. an Illumina platform and the GS Junior platform from Roche), the nucleic acid sequence of the lambda DNA in ERM-AD442k was verified. Non-lambda DNA sequences were identified in this material. The relative proportion of those sequences was estimated by NGS and further quantified by quantitative polymerase chain reaction (qPCR). Traces of the contaminating DNA, mainly coming from the Escherichia coli (E. coli) host used by Promega Corporation and Benelux BV to produce the lambda DNA, were negligible. Between-vial homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006. Within-vial homogeneity was quantified to determine the minimum sample intake. The certified DNA mass concentration value was obtained by ultraviolet (UV) spectrophotometry and the indicative DNA copy number concentration value by digital PCR (dPCR). The DNA copy number concentration measured by dPCR is consistent with the DNA mass concentration determined by UV spectrophotometry. The measurements were performed according to the scope of accreditation to ISO/IEC 17025:2005. The material was characterised by an inter-laboratory comparison exercise performed by laboratories of demonstrated competence and with adherence to ISO/IEC 17025. Technically invalid results were removed; however no other outliers were eliminated on statistical grounds only. Uncertainties of the certified and indicative values were calculated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) and include uncertainties relating to possible inhomogeneity and instability, and to characterisation. The material and its certified value are intended to be used for the calibration of DNA quantification methods, quality control and assessment of method performance. As any reference material, it can also be used to establish control charts or in validation studies. The indicative value of the material is, in contrast to the certified value, a value where the uncertainty was deemed too large to allow certification and is therefore less reliable than the certified value. The CRM is available in Axygen maximum recovery polypropylene vial containing a nominal volume of 1.1 mL lambda DNA in solution. The minimum amount of sample to be used is 50 µL for UV spectrophotometry and 68 µL for dPCR. The CRM was accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium.JRC.D.2-Standards for Innovation and sustainable Developmen

Certification of Plasmid DNA containing NK603 Maize DNA Fragments - Certified Reference Material ERM®-AD415

This report describes the preparation, characterisation, stability and suitability studies of the certified reference material (CRM) ERM-AD415 which contains a plasmid (pIRMM-0086) carrying a defined deoxyribonucleic acid (DNA) fragment specific for a genetic modification present in Zea Mays L. line NK603 as well as defined DNA fragments specific for the Zea mays taxon, of which one has been certified. The CRM was processed in 2009 and certified in 2010 by the European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), Geel, BEJRC.DG.D.2-Reference material

CERTIFICATION REPORT A set of three plasmid DNA calibration solutions bearing a porcine-specific DNA fragment Certified Reference Materials: ERM®-AD483a, ERM®-AD483b, ERM®-AD483c

This report describes the preparation and characterisation of a set of plasmid solutions, ERM-AD483a, ERM-AD483b and ERM-AD483c. The materials were produced in accordance with ISO Guide 34:2009 [1]. A DNA fragment specific for the identification of a porcine target was cloned into a pUC18 vector to construct the pIRMM-0104 plasmid. The nucleic acid sequence of the entire pIRMM-0104 plasmid was determined by dye terminator cycle sequencing. The plasmid was eluted in TElow buffer, and its concentration was measured by ultraviolet (UV) spectrophotometry. Afterwards, it was gravimetrically diluted to three different concentration levels. The plasmid copy number concentration of the three concentration levels were certified by digital and droplet digital quantitative polymerase chain reaction methods, dPCR and ddPCR respectively. In addition, between-unit homogeneity, as well as short-term and long-term stability was assessed in accordance with ISO Guide 35:2006 [2]. The materials are intended for the determination of a cut-off value to discriminate positive samples (containing the porcine target sequence) from negative samples by quantitative PCR as defined in the Standard Operating Procedure of the EU Reference Laboratory for Animal proteins (EURL-AP) according to Commission Regulation (EU) No 51/2013 [3, 4]. As any certified reference material (CRM), ERM-AD483 can also be used for control charts or validation studies. The CRM is available as a set of three vials, each containing at least 1 mL of plasmid solution. The minimum amount of sample to be used is 4 µL.JRC.D.2-Standards for Innovation and sustainable Developmen

A set of three plasmid DNA calibration solutions bearing a ruminant-specific DNA fragment Certified Reference Materials: ERM-AD482a,ERM-AD482b, ERM-AD482c

This report describes the preparation and characterisation of a set of three plasmid solutions, ERM-AD482a, ERM-AD482b and ERM-AD482c. The material was produced in accordance with ISO Guide 34:2009 [1]. A DNA fragment specific for the identification of ruminant meat was cloned into a pUC18 vector to construct the pIRMM-0103 plasmid. The nucleic acid sequence of the entire pIRMM-0103 plasmid was determined by dye terminator cycle sequencing applying the primer walking method on the entire plasmid. The plasmid was put into a solution and its concentration was measured by ultraviolet (UV) spectrophotometry. Afterwards this solution was gravimetrically diluted to obtain three different plasmid concentration levels. The plasmid copy number concentration of the three concentration levels were certified by digital quantitative polymerase chain reaction methods (dPCR). In addition, between-unit homogeneity, as well as short-term long-term and freeze-thaw stability was assessed in accordance with ISO Guide 35:2006 [2]. The materials are intended for the determination of a cut-off value to discriminate positive samples (containing the ruminant target sequence) from negative samples by quantitative PCR as defined in the Standard Operating Procedure of the EU Reference Laboratory for animal proteins (EURL-AP) according to Commission Regulation No 51/2013 [3, 4]. As any certified reference material (CRM), the materials can also be used for control charts or validation studies. The CRM is available as a set of three vials each containing at least 1 mL of plasmid solution. The minimum amount of sample to be used is 4 µL.JRC.D.2-Standards for Innovation and sustainable Developmen

Certification of Plasmid DNA containing 98140 Maize DNA Fragments - Certified Reference Material ERM®-AD427

This report describes the preparation, characterisation, stability and suitability of the certified reference material (CRM) ERM-AD427, which contains a plasmid (pIRMM-0090) carrying a defined 2'-desoxyribonucleic acid (DNA) fragment specific for a genetic modification present in Zea mays L. line 98140, as well as defined DNA fragment specific for the Zea Mays taxon. The CRM was processed in 2008 and certified in 2010 by the European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), Geel, BEJRC.DG.D.2-Reference material

The preparation and characterization of three solutions of plasmid DNA containing a ruminant-specific fragment with defined copy number concentrations - Reference Materials: IRMM-AD482a,IRMM-AD482b, IRMM-AD482c

This report describes the processing of a set of plasmid solutions, IRMM-AD482a, b and c. The material was produced following ISO Guide 34:2009. A DNA fragment specific for the identification of ruminant meat were cloned into a pUC18 vector to construct the pIRMM-0103 plasmid. The plasmid was diluted to three different concentration levels. Between unit-homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006. The materials are intended for the determination of a cut-off value to discriminate positive samples from negative samples by quantitative PCR. As any reference material, the materials can also be used for control charts or validation studies. The RM is available as a set of three plastic tubes containing 1 mL of plasmid solution. The minimum amount of sample to be used is 4 μL.JRC.D.2-Standards for Innovation and sustainable Developmen

Certification of Reference Materials of Maize Seed Powder containing Genetically Modified MON 810 Maize: Certified Reference Materials ERM®-BF413k (ERM®-BF413ak, ERM®-BF413ck, ERM®-BF413ek, ERM®-BF413gk)

The report describes the processing and certification of four maize seed powder Certified Reference Materials (CRMs) containing different mass fractions of genetically modified (GM) MON 810 maize (ERM-BF413ak, ck, ek, gk). The materials were processed and certified in 2007/2008 by the European Commission, Joint Research Centre, Institute for Reference Materials and Measurements IRMM, Geel, Belgium), according to the principles of ISO Guide 34. The four CRMs belonging to the ERM-BF413k series replace the earlier produced series ERM-BF413 which is exhausted. The four CRMs have been accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium.JRC.D.2-Reference material

CERTIFICATION REPORT The certification of different mass fractions of DP-ØØ4114-3 in maize seed powder Certified Reference Materials ERM®-BF439a, ERM®-BF439b, ERM®-BF439c, ERM®-BF439d and ERM®-BF439e

This report describes the production of a set of Certified Reference Materials (CRMs) ERM BF439a, b, c, d and e, which are certified for their 4114 maize event mass fractions. These materials were produced following ISO Guide 34:2009 and are certified in accordance with ISO Guide 35:2006. The materials are intended for the calibration or quality control of real-time PCR measurements to identify 4114 maize and/or quantify its mass fraction. As with any reference material, they can also be used for establishing control charts or for carrying out validation studies. The CRMs were accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium.JRC.D.2-Standards for Innovation and sustainable Developmen