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This report describes the production of two Certified Reference Materials (CRMs), ERM BF410dn and ERM-BF410gn, which are certified for their GTS 40-3-2 soya bean event mass fractions. These materials were produced following ISO Guide 34:2009 [1] and are certified in accordance with ISO Guide 35:2006 [2]. Genetically modified (GM) seeds of the GTS 40-3-2 soya bean event and seeds from a non-GM soya bean variety were milled to obtain GM and non-GM seed powders with a similar particle size distribution. Mixtures of non-GM and GM soya bean seed powder were prepared gravimetrically. The certified value was obtained from the gravimetric preparations, taking into account the genetic purity with respect to the GTS 40-3-2 soya bean event 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 [3]). 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 GTS 40-3-2 soya bean 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 soya bean seed powder, which were sealed under an atmosphere of argon. The minimum amount of sample to be used for extraction of the DNA 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 Development

Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to "absolute quantification", which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter. In addition, 2 orthogonal chemical-analysis methods based on nucleotide quantification, isotope-dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) were applied to quantify a more concentrated solution of the plasmid. Although 9 dPCR results from 8 laboratories showed some dispersion (relative standard deviation [RSD] = 11.8%), their means were closely aligned with those of the FCM-based counting method and the orthogonal chemical-analysis methods, corrected for gravimetric dilution factors. Using the means of dPCR results, the RSD of all 4 methods was 1.8%, which strongly supported the validity of the recent enumeration approaches. Despite a good overall agreement, the individual dPCR results were not sufficiently covered by the reported measurement uncertainties. These findings suggest that some laboratories may not have considered all factors contributing to the measurement uncertainty of dPCR, and further investigation of this possibility is warranted.

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 Development

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 Development

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 Development

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 Development

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 Development

This report describes the production of a set of Certified Reference Materials (CRMs) ERM BF437a, b, c, d and e, which are certified for their DAS-81419-2 mass fractions. This material was produced following ISO Guide 34:2009 and is certified in accordance with ISO Guide 35:2006. Genetically modified (GM) seeds of the soya event DAS-81419-2 and seeds from a non-GM soya variety were milled to obtain GM and non-GM seed powders with a similar particle size distribution. Mixtures of non-GM and GM soya powder were prepared gravimetrically. The certified value was obtained from the gravimetric preparations, taking into account the genetic purity with respect to the event DAS-81419-2 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). Uncertainties of the certified values were calculated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) 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 DAS-81419-2 soya 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 vials containing at least 1 g of dried soya seed powder which were sealed under atmosphere of argon. The minimum amount of sample to be used 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 Development

The content of this manual is based on the training course that was organised on the premises of the European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (Geel, BE) at the end of 2013. The training manual complements the training course that was intended to improve the quality of measurement results obtained when quantifying genetically modified organisms (GMO) in food and feed. Both, the training course and this manual, were developed in line with the current EU GMO legislation. The manual is addressed to laboratory managers and practitioners in analytical laboratories who perform GM quantification measurements and use reference materials for calibration, quality control and method validation including in-house verification. It is also intended for analysts who need to assess measurement uncertainties as required by (EC) No 1829/2003, (EC) No 619/2011 and ISO/IEC 17025:2005. This training document has been written by JRC-IRMM upon request of the European Union Reference Laboratory for Genetically Modified Food and Feed (EURL-GMFF) to further improve the reporting of National Reference Laboratories (NRLs) nominated under Regulation (EC) No 882/2004 and official GMO control laboratories within the EU. This manual is organised in four chapters covering the proper calibration of PCR methods, the estimation of measurement uncertainty, the establishment of metrological traceability of a measurement result and the way to prove the trueness of measurement results. The training manual is a didactic support of a previous guidance document that outlines issues related to the estimation of measurement uncertainty (MU) in the GMO sector. The training manual is also in line with the European technical guidance document for the flexible scope accreditation of laboratories quantifying GMOs, that is intended for laboratories that are acquiring or are holding a flexible scope of accreditation according to ISO/IEC 17025., JRC.D.2-Standards for Innovation and sustainable Development

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 Development

This report describes the production of the set of Certified Reference Materials (CRMs) ERM-BF434a, b, c, d and e. These are matrix materials certified for their 73496 rapeseed mass fractions. The material has been produced following ISO Guide 34:2009. Genetically modified (GM) rapeseeds of the event 73496 and of a non-GM rapeseed variety were ground to obtain GM and non-GM base powders. Gravimetric mixtures of non-GM and GM rapeseed powder were prepared by dry-mixing. The between unit-heterogeneity has been quantified and stability during dispatch and stability during storage have been assessed in accordance with ISO Guide 35:2006. The certified value was obtained from the gravimetric preparations, taking into account the purity of the base materials and their respective water mass fraction. The certified values were confirmed by event-specific real-time PCR as an independent verification method (measurements within the scope of accreditation to ISO/IEC 17025:2005). Uncertainties of the certified values were calculated in compliance with the Guide to the Expression of Uncertainty in Measurement (GUM) and include uncertainties related to possible inhomogeneity (Section 4), instability (Section 5) and characterisation (Section 6). The materials are intended for the calibration or quality control of 73496 rapeseed identification and quantification methods. As any reference material, they can also be used for control charts or validation studies. The CRMs are available in glass vials containing at least 1 g of dried rapeseed powder closed under argon atmosphere. The minimum amount of sample to be used is 200 mg. The CRM has been 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 Development

This report describes the production of a set of Certified Reference Materials (CRMs) ERM BF436a, b, c, d and e, certified for their DAS-44406-6 mass fractions. The material was produced following ISO Guide 34:2009. Genetically modified (GM) seeds of the soya event DAS-44406-6 and of a non-GM soya variety were milled to obtain GM and non-GM powders. Gravimetric mixtures of non-GM and GM soya powder were prepared by dry-mixing. Between-unit homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006. The certified value was obtained from the gravimetric preparations, taking into account the purity of the base materials and their water mass fraction. The certified values were confirmed by event-specific real-time PCR as independent verification method (measurements within the scope of accreditation to ISO/IEC 17025:2005). Uncertainties of the certified values were calculated in compliance with the Guide to the Expression of Uncertainty in Measurement (GUM) and include uncertainties related to possible inhomogeneity (Section 4), instability (Section 5) and characterisation (Section 6). The materials are intended for the calibration or quality control of methods. As any reference material, they can be used for establishing control charts or validation studies. The CRMs are available in glass vials containing at least 1 g of dried soya seed powder and closed under argon atmosphere. The minimum amount of sample to be used 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 Development

The aim of this guidance document is to facilitate harmonised flexible scope accreditation within Europe, according to ISO/IEC 17025:2005 related to quantitative testing of genetically modified organisms (GMOs) by quantitative real-time polymerase chain reaction (qPCR) for GM events authorised in the EU or which are in the authorisation process. This document gives guidance to and is intended for laboratories that are considering to acquire a flexible scope of accreditation according to ISO/IEC 17025. At the same time it aims to provide information for assessors involved in the accreditation process of these laboratories. This guidance document has been written by members of the Task Force (TF) Flexible scope accreditation, which has been initiated by European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (EC JRC-IRMM, Geel, BE). After an extensive commenting phase it has been submitted to the European co-operation for Accreditation (EA) in February 2013 for consideration as an EA guidance document., JRC.D.2-Standards for Innovation and sustainable Development

This report describes the production of a set of Certified Reference Materials (CRMs) ERM-BF431a, b, c, d and e, matrix materials certified for their AV43-6-G7 mass fractions. The material has been produced following ISO Guide 34:2009. Genetically modified (GM) tubers of the potato event AV43-6-G7 and of a non-GM potato variety were dried and ground to obtain GM and non-GM potato powders. Beside these two pure materials gravimetric mixtures of non-GM and GM potato powder were prepared by dry-mixing. Between unit-heterogeneity has been quantified and stability during dispatch and storage have been assessed in accordance with ISO Guide 35:2006. The certified value was obtained from the gravimetric preparations, taking into account the purity of the base materials and their water mass fraction. The certified values were confirmed by event-specific real-time PCR as independent verification method (measurements within the scope of accreditation to ISO/IEC 17025:2005). Uncertainties of the certified values were calculated in compliance with the Guide to the Expression of Uncertainty in Measurement (GUM) and include uncertainties related to possible heterogeneity, instability, and characterisation. The materials are intended for the calibration or quality control of methods. As any reference material, they can also be used for control charts or validation studies. The CRMs are available in glass vials containing at least 1 g of dried potato tuber powder and closed under argon atmosphere. The minimum amount of sample to be used is 200 mg. The CRM has been 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 Development

This report describes the production of a set of Certified Reference Materials (CRMs) ERM-BF433a, b, c and d, matrix materials certified for their DAS-40278-9 mass fractions. The material has been produced following ISO Guide 34:2009. Genetically modified (GM) seeds of the maize event DAS-40278-9 and of a non-GM maize variety were ground to obtain GM and non-GM base powders. Gravimetric mixtures of non-GM and GM maize powder were prepared by dry-mixing. Between unit-heterogeneity has been quantified and stability during dispatch and storage have been assessed in accordance with ISO Guide 35:2006. The certified value was obtained from the gravimetric preparations, taking into account the purity of the base materials and their respective water mass fraction. The certified values were confirmed by event-specific real-time PCR as independent verification method (measurements within the scope of accreditation to ISO/IEC 17025:2005. Uncertainties of the certified values were calculated in compliance with the Guide to the Expression of Uncertainty in Measurement (GUM) and include uncertainties related to possible heterogeneity, instability, and characterisation. The materials are intended for the calibration or quality control of methods. As any reference material, they can also be used for control charts or validation studies. The CRMs are available in glass vials containing at least 1 g of dried maize seed powder, closed under argon atmosphere. The minimum amount of sample to be used is 200 mg. The CRM has been 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 Development

This report describes the production of a set of plasmid solutions, ERM®-AD623a, b, c, d, e, and f, certified for the copy number ratio of specific desoxyribonucleic acid (DNA) fragments per plasmid and for the copy number concentration of the plasmid. The plasmid contains a single copy of 3 DNA fragments: one specific for the transcript of breakpoint cluster region gene (BCR), one specific for the transcript of the glucuronidase, beta gene (GUSB) and one specific for the fusion transcript from the BCR gene and the c-abl oncogene 1 (BCR-ABL)., JRC.D.2-Standards for Innovation and sustainable Development

This report describes the production of a set of Certified Reference Materials (CRMs) ERM-BF432a, b, c and d, matrix materials certified for their DAS-68416-4 mass fractions. The material has been produced following ISO Guide 34:2009 [1].Genetically modified (GM) seeds of the soya event DAS-68416-4 and of a non-GM soya variety were ground to obtain GM and non-GM base powders. A non-GM pure material was prepared and gravimetric mixtures of non-GM and GM soya powder were prepared by dry-mixing. Between unit-heterogeneity has been quantified and stability during dispatch and storage have been assessed in accordance with ISO Guide 35:2006 [2].The certified value was obtained from the gravimetric preparations, taking into account the purity of the base materials and their water mass fraction. The certified values were confirmed by event-specific real-time PCR as independent verification method (measurements within the scope of accreditation to ISO/IEC 17025).Uncertainties of the certified values were calculated in compliance with the Guide to the Expression of Uncertainty in Measurement (GUM) [3] and include uncertainties related to possible heterogeneity and instability and to characterisation. The materials are intended for the calibration or quality control of methods. As any reference material, they can also be used for control charts or validation studies. The CRMs are available in glass vials containing 1 g of dried soya seed powder closed under argon atmosphere. The minimum amount of sample to be used is 500 mg.The CRM has been 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 Development

The provision of traceable standards to the biological community is an area of active research in many NMIs.The quantification of the relative amount of DNA sequences extracted from a biological tissue remains a complex analytical procedure and relies on the availability of such standards. Real-time PCR is currently the most applied measurement method to identify and quantify DNA sequences. Several NMIs were able to demonstrate their ability to use this technology to quantify a defined plasmid DNA using the same plasmid DNA as a calibrant (CCQM-P44 (1&2) and CCQM KC-61). The goal of this CCQM 113 pilot study was to demonstrate the ability to quantify DNA sequences present in a biological tissue using an independent calibration system. The quantification has been performed by QRT-PCR. One laboratory has performed an absolute quantification of both targets by digital PCR without the use of an external standard on sample 1 and found precisely the expected GM concentration. This result reveals the potential of such new technology for absolute quantification of DNA targets. The samples 1 and 3 had a very low number of 1507 targets which has created some difficulties for several laboratories as the unknown samples were too diluted and falling outside calibration curves. The data for the undiluted samples have nevertheless been reported here. Most participating NMIs managed to quantify the 4 samples with high accuracy and reported measurements very close to the assigned value. They could clearly demonstrate their ability to quantify DNA sequences in a biological tissue., JRC.DDG.D.2-Reference materials

The potential approaches for third-party assessment of reference material producers are revisited and the activities of the Reference Materials (RM) Unit of the Institute for Reference Materials and Measurements (IRMM) to obtain accreditation to ISO Guide 34 and ISO 17025 are described. Accreditation was achieved for the Unit, as all matrix RM activities of the institute are concentrated there. A management system was established that allows suffiecient freedom to be applicable to a wide range of RMs while being precise enough to ensure compliance with ISO Guides 30, 31 and especially 34 and 35. Accreditation was achieved in 2004 with independent scopes for testing and RM production and was confirmed and extended in 2005. The key point of the RM Unit management system for RM production are given., JRC.D.2-Reference materials

A candidate digital PCR (dPCR)-based reference measurement procedure for quantification of human cytomegalovirus (hCMV) was evaluated in 10 viral load comparison schemes (seven external quality assessment (EQA) and three additional training schemes) organized by INSTAND e.V. over four years (between September 2014 and March 2018). Four metrology institutes participated in these schemes using the same extraction method and dPCR measurement procedure for the hCMV specific target sequence of UL54 gene. The calibration independent reference measurement procedure results from the metrology institutes were compared to the results of the clinical diagnostic laboratories applying hCMV qPCR measurement procedures calibrated to reference materials. While the criteria for the acceptable deviation from the target value interval for INSTAND's EQA schemes is from-0.8 log10 to +0.8 log(10), the majority of dPCR results were between-0.2 log10 to +0.2 log(10). Only 4 out of 45 results exceeded this interval with the maximum deviation of-0.542 log(10). In the training schemes containing samples with lower hCMV concentrations, more than half of the results deviated less than +/- 0.2 log(10) from the target value, while more than 95% deviated less than +/- 0.4 log(10) from the target value. Evaluation of intra-and inter-laboratory variation of dPCR results confirmed high reproducibility and trueness of the method. This work demonstrates that dPCR has the potential to act as a calibration independent reference measurement procedure for the value assignment of hCMV calibration and reference materials to support qPCR calibration as well as ultimately for routine hCMV load testing. This study was financially supported by the INFECTMET project (the EMRP project; jointly funded by the EMRP participating countries within EURAMET and the European Union, project no. HLT08); AntiMicroResist project (which has received funding from the EMPIR program co-financed by the Participating States and from the European Union’s Horizon 2020 research and innovation program, project no. 15HLT07); the Slovenian Research Agency (contract no. P4-0165 and 1000-13-0105); Ministry of Education, Science and Sport of republic of Slovenia (contract no. 3330-14-500133) and supported by the UK National Measurement System. The dPCR equipment used at NIB in this study was financed by the Metrology Institute of the Republic of Slovenia (MIRS), with financial support from the European Regional Development Fund. The funding source(s) had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. This publication reflects only the authors’ s views and EURAMET is not responsible for any use that may be made of the information it contains.

The European Union Reference Laboratory for Genetically Modified Food and Feed (EURL GMFF) organised a proficiency test (PT) for National Reference Laboratories (NRLs) to support the official controls on food and feed in line with Regulation (EC) No 882/2004. Other official control laboratories were allowed to participate on a voluntary basis. Two test items were distributed: test item 1 (T1) was composed of ground chicken feed spiked with a mixture of GM soybean event MON-Ø4Ø32-6 (40-3-2) and non-GM soybean, and test item 2 (T2) was a soybean flour containing the same GM soybean event 40-3-2. Participants were required to screen T1 and T2 for the presence of three GM soybean events, and to quantify the event that was present with the highest GM mass fraction. The results had to be reported in GM mass fraction (mass/mass %). Eighty-six participants from 39 countries participated to this PT, including 54 NRLs, of which 33 are designated in line with Regulation (EC) No 882/2004 (NRL/882) and 21 are nominated in Regulation (EU) No 120/2014 to support the EURL GMFF on method validation (NRL/120), as well as 10 other EU official control laboratories. The qualitative results, i.e. the correct identification of the GM event, were evaluated and scored as correct or incorrect. The assigned value for the 40-3-2 soybean mass fraction in both test materials was derived as the robust mean of the data provided by the NRLs. Laboratory performance was primarily evaluated by calculating z scores. The results reported indicate that all participants identified the correct GM event in both test items. All, but one NRL obtained a satisfactory performance (z) score for the reported 40-3-2 soybean mass fraction in both test items and the performance of one other NRL was unacceptable for T1 because they reported the 40-3-2 mass fraction as below their LOQ. Six and two NRLs obtained a questionable z score for T1 and T2, respectively. Considering the results provided by the other participants (non-NRL), three and two non-NRLs obtained an unsatisfactory z score for T1 and T2, respectively. Another three laboratories obtained a questionable z score for T1 and one laboratory for T2. The laboratories' ability to provide results close to the assigned value within their claimed measurement uncertainty was additionally evaluated by ζ scores. Twenty four and 16 laboratories had an unsatisfactory ζ scores for T1 and T2, respectively. Unsatisfactory ζ scores were mainly the consequence of an underestimated or not reported measurement uncertainty. Guidance is provided for correctly estimating the measurement uncertainty of analytical results. A root-cause analysis will be requested from NRLs with an unsatisfactory z score result in this PT and will be followed-up., JRC.F.5-Food and Feed Compliance