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

Verification of analytical methods for GMO testing when implementing interlaboratory validated methods: Version 2

In the EU, method validation is an essential part of the process that regulates the introduction of new GMOs as food and/or feed into the market. When the inter-laboratory validation study is completed, the method is ready to be implemented in routine testing laboratories. When implementing the new method, the laboratory has to verify that the method can be used for its intended purpose (method verification). The scope of this document is to provide guidance on how to carry out the method verification of inter-laboratory validated methods for the qualitative and quantitative detection of GMOs. Considering that the Polymerase Chain Reaction (PCR) is the method of choice in the EU for the identification and quantification of GMOs, this document refers exclusively to real time PCR. However, if novel methods are subsequently developed that fulfil legal requirements, then this document will be amended accordingly.JRC.F.5-Food and Feed Complianc

New approaches in GMO detection

The steady rate of development and diffusion of GM plants and their increasing diversification in characteristics, genes and genetic control elements poses a challenge in GMO analysis. It is expected that in the near future the picture will be even more complex. Traditional approaches, mostly based on the sequential detection of one target at a time, or on a limited multiplexing, allowing only few targets to be analysed at once, do no longer meet the testing requirements. Along with the development of new analytical technologies, new approaches towards global GMO analysis have been developed that rely on 1) a smart and accurate strategy for target selection, 2) the use of high throughput systems or platforms for the detection of multiple targets and 3) on algorithms that allow the conversion of analytical results into an indication of the presence of individual GMO(s) potentially present in an unknown sample. This paper addresses the critical aspects listed above taking examples from the most recently developed strategies and tools.JRC.DG.I.4-Molecular biology and genomic

Application of whole genome shotgun sequencing for detection and characterization of genetically modified organisms and derived products

The emergence of high-throughput, massive or next-generation sequencing technologies has created a completely new foundation for molecular analyses. Various selective enrichment processes are commonly applied to facilitate detection of predefined (known) targets. Such approaches, however, inevitably introduce a bias and are prone to miss unknown targets. Here we review the application of high-throughput sequencing technologies and the preparation of fit-for-purpose whole genome shotgun sequencing libraries for the detection and characterization of genetically modified and derived products. The potential impact of these new sequencing technologies for the characterization, breeding selection, risk assessment, and traceability of genetically modified organisms and genetically modified products is yet to be fully acknowledged. The published literature is reviewed, and the prospects for future developments and use of the new sequencing technologies for these purposes are discussed

How to Reliably Test for GMOs

Accurate and reliable testing is necessary to support the requirements of legislation on genetically modified organisms (GMO) defining their traceability and labelling. In this book, an overview of all key topics relevant to GMO testing is presented, including practical experience and generally accepted laboratory practices. GMO legislation, sources of information on GMOs, organization of the testing laboratory focusing on aspects of quality system and methods for testing are described. Additionally, precise information on qualitative and quantitative real-time PCR detection with special attention to critical points and important precautionary measures to assure reliable and accurate analyses are given. Special attention was given also to metrological topics, such as validation and verification of methods and measurement uncertainty. The approaches for GMO detection, which are precisely described in the present document, are also relevant for other areas where detection and identification rely on nucleic acid-based methods. Numerous diverse GMOs are coming onto the market including GMOs produced by new technologies, which are challenging established systems of analysis, therefore new developments in detection technologies and bioinformatics solutions are presented.JRC.I.4-Molecular biology and genomic

Knowledge-technology-based discovery of unauthorized genetically modified organisms

Various legislations worldwide provide a framework for the authorization of genetically modified organisms (GMOs) and products derived therefrom [1]. Despite these regulations, novel GMOs occasionally enter the market without authorization [2¿22] (Table 1) and are referred to as unauthorized GMOs (UGMs). The presence of UGMs in food and feed raises safety and labeling concerns, and challenges international trade [23, 24]. Recently reported UGM incidents have created an urgent need to harmonize regulations at a global level, and call for appropriate strategies to discover UGMs. However, some novel UGMs are intrinsically difficult to detect using current analytical strategies for reasons outlined herein. We therefore propose a paradigm shift in the way UGMs can be discovered: a documentationbased screening for products that potentially contain UGMs using knowledge technologies, followed by analytical confirmation. Here, we will describe the main concepts of the novel approach, illustrate it with a case study, and outline benefits, limitations, and complementarities compared with the current analytical detection strategy.JRC.DDG.I.4-Molecular biology and genomic

DNA enrichment approaches to identify unauthorized genetically modified organisms (GMOs)

With the increased global production of different genetically modified (GM) plant varieties, chances increase that unauthorized GM organisms (UGMOs) may enter the food chain. At the same time, the detection of UGMOs is a challenging task because of the limited sequence information that will generally be available. PCR-based methods are available to detect and quantify known UGMOs in specific cases. If this approach is not feasible, DNA enrichment of the unknown adjacent sequences of known GMO elements is one way to detect the presence of UGMOs in a food or feed product. These enrichment approaches are also known as chromosome walking or gene walking (GW). In recent years, enrichment approaches have been coupled with next generation sequencing (NGS) analysis and implemented in, amongst others, the medical and microbiological fields. The present review will provide an overview of these approaches and an evaluation of their applicability in the identification of UGMOs in complex food or feed samples

Method Validation and Quality Management in the Flexible Scope of Accreditation: an Example of Laboratories Testing for Genetically Modified Organisms

Quality assurance is a prerequisite for accurate and reliable results in food and feed testing, ISO/IEC 17025 being recognized worldwide as the base standard. A flexible scope of accreditation enables testing laboratories to react quickly to customer demand and to cope with the large number of new methods, which have to be introduced in the laboratory. Precisely defined procedures for the validation of methods, together with performance and acceptance criteria, are the key points for flexible scope of accreditation. Testing for genetically modified organisms (GMO) is a challenging exercise, especially with more GMOs entering the world market. We describe here the organization and performance of validating quantitative detection methods for GMO testing in the context of the EU legislation. Operational procedures for method validation organized by the Community Reference Laboratory for Genetically Modified Food and Feed (CRL-GMFF), assisted by the European Network of GMO Laboratories (ENGL) are described. A protocol for validating methods within an individual laboratory is proposed and discussed in terms of the requirements of flexible scope of accreditation. The system setup can be an example for other similar fields of analytical work.JRC.I.6-Biotechnology and GMO

Metabolic Discrimination of Catharanthus roseus Leaves Infected by Phytoplasma Using (1)H-NMR Spectroscopy and Multivariate Data Analysis

A comprehensive metabolomic profiling of Catharanthus roseus L. G. Don infected by 10 types of phytoplasmas was carried out using one-dimensional and two-dimensional NMR spectroscopy followed by principal component analysis (PCA), an unsupervised clustering method requiring no knowledge of the data set and used to reduce the dimensionality of multivariate data while preserving most of the variance within it. With a combination of these techniques, we were able to identify those metabolites that were present in different levels in phytoplasma-infected C. roseus leaves than in healthy ones. The infection by phytoplasma in C. roseus leaves causes an increase of metabolites related to the biosynthetic pathways of phenylpropanoids or terpenoid indole alkaloids: chlorogenic acid, loganic acid, secologanin, and vindoline. Furthermore, higher abundance of Glc, Glu, polyphenols, succinic acid, and Suc were detected in the phytoplasma-infected leaves. The PCA of the (1)H-NMR signals of healthy and phytoplasma-infected C. roseus leaves shows that these metabolites are major discriminating factors to characterize the phytoplasma-infected C. roseus leaves from healthy ones. Based on the NMR and PCA analysis, it might be suggested that the biosynthetic pathway of terpenoid indole alkaloids, together with that of phenylpropanoids, is stimulated by the infection of phytoplasma

Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA

Quantitative PCR (qPCR) is an important tool in pathogen detection; however, the use of different qPCR components, calibration materials and DNA extraction methods reduces the comparability between clinics, which could result in false diagnosis and discrepancies in patient care. The establishment of a metrological framework for nucleic-acid tests is expected to improve the degree of standardisation of pathogen detection and quantification methods applied in a clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) allows quantification of nucleic acids and has already been used for a myriad of applications, including pathogen quantification. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessments on its actual performance characteristics should be collected before it can be implemented in a metrological framework and to allow an adequate estimation of the measurement uncertainty. Here, high repeatability and reproducibility of dPCR for quantification of DNA from human cytomegalovirus were demonstrated. Using extracted DNA and whole-virus material, each of five dPCR platforms from four laboratories demonstrated high intermediate precision between three consecutive experiments. Furthermore, discrepancies in estimated mean DNA copy-number concentrations between different laboratories were less than two-fold, with DNA extraction recognised as the main source of variability. Our results demonstrate dPCR-based methods can be very repeatable and reproducible for quantification of viral DNA, and should be considered as potent reference method candidates for implementation in a metrological framework.JRC.F.6-Reference Material