Training manual on GMO quantification: Proper calibration and estimation of measurement uncertainty

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 Developmen

Guidance Document on Measurement Uncertainty for GMO Testing Laboratories

The report outlines the technical issues related to the estimation of measurement uncertainty (MU) involved in the GMO sector. In particular it gives guidance to GMO testing laboratories how to estimate the analytical variability of quantitative analytical results obtained by real-time PCR. Two selected approaches for the estimation of MU are presented in detail; references to alternative approaches are given. The first approach are given. The first approach presented in detail is using data from collaborative trial in combination with in-house quality control data for the estimation of MU. An alternative approach using data from within-laboratory samples for the estimation of MU is presented.JRC.D.2-Reference material

Certification of Plasmidic DNA containing MON 810 Maize DNA fragments, Certified Reference Materials ERM-AD413

This report describes the preparation, homogeneity, stability and certification studies of a plasmidic deoxyribonucleic acid (DNA) (ERM-AD413) containing a defined DNA fragment specific for a genetic modification present in Zea Mays MON 810 event (1) as well as a defined DNA fragment specific for the Zea Mays taxon. The CRM was processed in 2005 and certified in 2007 by the European Commission, Directorate General Joint Research Centre, Institute for Reference Materials and Measurements (IRMM) in Geel, Belgium. The CRM is available in plastic tubes containing 500 µL of the plasmidic DNA in 1 mmol/L EDTA pH 7.0 buffer. Each tube contains approximately 10 to the power of 9 copies of the ERM-AD413 plasmid which correspond approximately to 3.41 ng of DNA. The plasmid contains a 170 bp fragment of the MON 810 5' plant-P35S junction and a 351 bp fragment of the maize endogenous high mobility group gene (hmg) (2). both DNA sequences originated from DNA extracted from MON 810 seeds supplied by RAGT Semences (Rodez, France). The certified value is the number of each cloned DNA fragments per plasmid. The number ratio between those two DNA fragments is given as an indicative value measured by duplex and simplex real-time PCR.JRC.D.2-Reference material

CERTIFICATION REPORT: The certification of the catalytic activity concentration of alpha-amylase in ERM® AD456/IFCC

This report describes the production of ERM®-AD456/IFCC, which is a material certified for the catalytic activity concentration of alpha-amylase. This material was produced following ISO 17034:2016 and is certified in accordance with ISO Guide 35:2017. The starting material was pancreatic alpha-amylase purified from human tissue. The enzyme was diluted in a buffered solution which was filled into glass vials and lyophilised. Between unit-homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2017. The material was characterised by an interlaboratory comparison of laboratories of demonstrated competence and adhering to ISO/IEC 17025. Uncertainties of the certified values were calculated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) and include uncertainties related to possible inhomogeneity, instability and characterisation. The material is intended for the assessment of method performance of the primary reference measurement procedure (PRMP) for the catalytic activity concentration of alpha–amylase at 37 °C established by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) [ , ]. In addition the material can also be used as trueness control or external quality control material for routine measurement systems if commutability has been proven for the assay concerned. As with any reference material, it can be used for establishing control charts or in validation studies. The CRM is available in glass vials containing lyophilised material from 1 mL of alpha-amylase solution which were sealed under an atmosphere of nitrogen. The minimum amount of sample to be used is 5 µL after reconstitution of the whole content of 1 vial in 1 mL.JRC.F.6-Reference Material

Certification of Reference Materials of Cotton Seed Powder with Different Mass Fractions of the Cotton Event T304-40 - Certified Reference Materials ERM®-BF429 (ERM®-BF429a, ERM®-BF429b, ERM®-BF429c)

This report describes the processing and certification of three cotton seed powder Certified Reference Materials (CRMs) containing different mass fractions of genetically modified (GM) T304-40 cotton (ERM-BF429a, b, c). The materials were prepared and certified in 2009/2010 by the European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM, Geel, Belgium), according to the principles of ISO Guide 34.JRC.DG.D.2-Reference material

The certification of different mass fractions of MON-Ø4Ø32-6 in soya bean powder Certified Reference Materials ERM®-BF410ap, ERM®-BF410bp, ERM®-BF410cp, ERM®-BF410dp and ERM®-BF410ep

This report describes the production of a set of Certified Reference Materials (CRMs), ERM BF410ap, bp, cp, dp and ep, which are certified for their GTS 40-3-2 soya bean (unique identifier MON-Ø4Ø32-6) 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 GTS 40-3-2 soya bean and 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 panel.JRC.F.6-Reference Material

Guidance document on Measurement Uncertainty for GMO Testing Laboratories 3rd Edition

This document provides guidance to GMO control laboratories on how to estimate measurement uncertainty (MU) associated with any GMO measurement result. Competent authorities have a responsibility to decide if a measurement result exceeds the legal threshold. Consequently control laboratories must estimate and report the MU of their measurement results. Quantitative polymerase chain reaction (qPCR) and digital polymerase chain reaction (dPCR) are the methods applied for the enforcement of EU law on GMO labelling. This document takes into account: current EU legislation, the availability of certified reference materials (CRMs) and validated methods, and the need for control laboratories which conduct measurements for the enforcement of EU legislation to be accredited according to ISO/IEC 17025. Based on these, two approaches for the estimation of MU for PCR measurement results are presented in this guidance document. References to alternative approaches are also provided.JRC.F.5-Food and Feed Complianc

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