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Guidance Document on Measurement Uncertainty for GMO Testing Laboratories
... The uncertainty of real-time PCR measurements is primarily composed of two components, calibrator uncertainty and measurement process uncertainty; both these components are assumed to follow a normal distribution 14,15 . Therefore, measurement results are represented by the mean DNA concentration of each sample and symmetrical measurement uncertainty. ...
... Mean contaminant DNA copy number in a well from reagents and environment (13) n new Estimate of the expectation of N new (14) u(n new ) Symmetric uncertainty of n new (14) σ N_new Standard deviation of N new (14) A n_new , B n_new Left and right intervals for representing the asymmetric variety of N new (15) and (16) A n_new , B n_new Left and right confidence intervals for the expectation of N new (15) and (16) u(e curve ) Symmetric uncertainty of a calibration curve in real-time PCR process (17) e curve Estimation of the error of the mean quantification result sourced from the calibration curve (17) n Q Mean PCR quantification result of replicates of target samples (17) N rw,i Effective number of wells containing calibrators with the same i-th representative DNA copy number (17) n r,i Estimate of mean DNA copy number of calibrators with the same i-th representative DNA copy number (17) R n_r,i Representative value of n r,i ...
... Mean contaminant DNA copy number in a well from reagents and environment (13) n new Estimate of the expectation of N new (14) u(n new ) Symmetric uncertainty of n new (14) σ N_new Standard deviation of N new (14) A n_new , B n_new Left and right intervals for representing the asymmetric variety of N new (15) and (16) A n_new , B n_new Left and right confidence intervals for the expectation of N new (15) and (16) u(e curve ) Symmetric uncertainty of a calibration curve in real-time PCR process (17) e curve Estimation of the error of the mean quantification result sourced from the calibration curve (17) n Q Mean PCR quantification result of replicates of target samples (17) N rw,i Effective number of wells containing calibrators with the same i-th representative DNA copy number (17) n r,i Estimate of mean DNA copy number of calibrators with the same i-th representative DNA copy number (17) R n_r,i Representative value of n r,i ...
Recently, in food safety and various other fields, qualitative and quantitative gene analysis using real-time polymerase chain reaction (PCR) method has become increasingly popular. The limit of detection (LOD) and quantifiable range for these measurements depends on the range and precision of DNA calibrators’ concentrations. Low-copy-number nucleic acid reference materials with low uncertainty produced by an inkjet system have been developed to allow for precise measurements in a low-copy-number region. However, when using a calibrator with a low copy number near one, the copy number distribution is asymmetric. Consequently, the confidence intervals of estimated copy numbers can include negative values when conventional methods of uncertainty estimation are used. A negative confidence interval is irrelevant in the context of copy number, which is always positive value or zero. Here, we propose a method to evaluate the uncertainty of real-time PCR measurements with representative values and an asymmetric 95% confidence interval. Moreover, we use the proposed method for the actual calculation of uncertainty of real-time PCR measurement results for low-copy-number DNA samples and demonstrate that the proposed method can evaluate the precision of real-time PCR measurements more appropriately in a low-copy-number region.
... In this approach, data from collaborative trials and sample analyses, including all the factors infl uencing the MU during the analytical procedure, are used as a source for estimation of MU. This approach is described in detail in the guidance document on measurement uncertainty for GMO testing laboratories produced by the ENGL working group on measurement uncertainty (Trapmann et al. 2009 ) . ...
... At the time the ENGL guidance document on measurement uncertainty for GMO testing laboratories was published, there were not enough data obtained from collaborative trials to calculate a common value that could be used as an estimate of the MU in GMO testing based on collaborative trials (Trapmann et al. 2009 ) . ...
... Using the data from Kodama and collaborators, assuming the absence of bias and that the absolute uncertainty u o is negligible, the standard uncertainty u can be approximated by S R as described in equation 12 of the ENGL guidance document on measurement uncertainty for GMO testing laboratories (Trapmann et al. 2009 ) . ...
The detection of genetically modified organisms (GMOs) is becoming very complex, with new GMOs, approved and unapproved, constantly entering world markets. Traceability and labelling of GMOs is defined in regulations worldwide, demanding accurate and reliable testing to support the requirements of legislation. This Brief provides the current state-of-the-art on all key topics involved in GMO testing and is a source of detailed practical information for laboratories. Special focus is given to qualitative and quantitative real-time PCR analysis relevant to all areas where detection and identification rely on nucleic acid-based methods. The following topics, important for testing laboratories, are also discussed: organization of the laboratory, focusing on aspects of the quality system and methods for testing, validation and verification of methods, and measurement uncertainty. The Brief also discusses the new challenges of GMOs and novel modified organisms, using new technologies, and the possible solutions for GMO detection, including bioinformatics tools. Finally, legislation on GMOs and sources of information on GMOs are provided, which are relevant not only to testing laboratories, but to anyone interested in GMOs. The authors of this Brief have many years of experience in GMO testing, development of real-time PCR methods, implementation of quality system requirements, validations and verification of methods, and measurement uncertainty. The National Institute of Biology is a highly qualified research laboratory and a National Reference Laboratory, which also performs routine analyses of food, feed and seed. The Institute for Health and Consumer Protection of the European Union Joint Research Centre has extensive knowledge and experience of GMO detection. It hosts the European Union Reference Laboratory for GM Food and Feed in addition to chairing the European Network of GMO Laboratories. © Jana Žel, Mojca Milavec, Dany Morisset, Damien Plan, Guy Van den Eede, Kristina Gruden 2012. All rights reserved.
... For routine application in the scope of ISO 17025 full validation has to be done to show the suitability of such methods. Several guidelines were released for the validation of PCR systems already [10][11][12]. Although they are an important help to resolve the different aspects during each validation step, they remain unclear in the concrete steps and details and do not address digital PCR specifically. ...
... We validated 13 droplet digital PCR systems according to guidelines [10][11][12], fulfilling ISO17025 requirements using certified reference material. The lower range of quantification could be confirmed for all systems at a level of 0.1% GMO. ...
... For maize this factor varied significantly. The measurement uncertainty was calculated by geometrical addition of SD and accuracy [11]. MU for the ddPCR™ and real time PCR are compiled, MU with Bold were superior (difference >1%) a An earlier publication [7] observed for a 1% reference material a copy number ratio of 0.395. ...
Digital PCR methods were recently introduced in food analysis. To run these methods within the scope of ISO17025, they have to be validated. Although several guidelines are available, each laboratory has to implement these guidelines in an adapted validation scheme. We present here one possible implementation. We chose 13 GMO traits which were predominantly detected in the past. The results show that in the range of 1% GMO content, the digital PCR has a little superior performance compared to real time PCR. In the range of the detection limit, measurement uncertainty remains comparable to real time PCR. During validation, a conversion factor was determined for each trait suggesting that the calculation from % copies/copies to % weight/weight may be possible. This shows that GMO contents can be measured without the use of reference material by the validation described here and determination of a conversion factor, which is a great improvement in terms of expense and storage capacity.
... In this approach, data from collaborative trials and sample analyses, including all the factors infl uencing the MU during the analytical procedure, are used as a source for estimation of MU. This approach is described in detail in the guidance document on measurement uncertainty for GMO testing laboratories produced by the ENGL working group on measurement uncertainty (Trapmann et al. 2009 ) . ...
... At the time the ENGL guidance document on measurement uncertainty for GMO testing laboratories was published, there were not enough data obtained from collaborative trials to calculate a common value that could be used as an estimate of the MU in GMO testing based on collaborative trials (Trapmann et al. 2009 ) . ...
... Using the data from Kodama and collaborators, assuming the absence of bias and that the absolute uncertainty u o is negligible, the standard uncertainty u can be approximated by S R as described in equation 12 of the ENGL guidance document on measurement uncertainty for GMO testing laboratories (Trapmann et al. 2009 ) . ...
Accurate and reliable testing is necessary to support the requirements of legislation on genetically modified organisms (GMO) defining their traceability and labeling. 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 the quality system, and methods for testing are described. In addition, 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.
... The difficulty can be easily understood as there is a lack of coherence between the EU legal requirements and the approaches followed by laboratories when performing the measurement [9,10]. The estimation of uncertainty associated with the measurement result remains a difficult topic as noticed during the comparative testing campaigns organised by the EURL-GMFF, despite the fact that a number of guidance documents and specific applications notes have been published to help to correctly implement the GMO legislation in Europe [5,6,11,12]. Therefore, the Steering Committee of the European Network of GMO Laboratories (ENGL) asked the JRC-IRMM to organize a training course that should tackle those challenging topics. ...
... The second approach uses data from routine measurements. Estimations are made of a constant uncertainty contribution associated with measurements at the limit of quantification (LOQ) (u 0 ), and of a proportional, concentration-dependent uncertainty contribution associated with measurements above the LOQ (u var,rel ; in other documents called RSU [5] or u pro,bias,rel [6]). The two standard uncertainties u 0 and u var,rel are summed up to obtain the combined standard uncertainty (u c ): ...
... Also for this approach, detailed calculations are given in a practical example in Annex II (2.2). Besides, it has been described in detail elsewhere [5,6]. ...
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 [1,2].
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
[1], (EC) No 619/2011 [2] and ISO/IEC 17025:2005 [3].
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 [4] 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 [5]. 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 [6].
... Method performance criteria such as the within-laboratory reproducibility standard deviation (S R ) and the within-laboratory reproducibility relative standard deviation (RSD R ), limit of detection (LOD), limit of quantification (LOQ), and linearity were determined for each RT-qPCR assay based on the Guidance Document on Measurement Uncertainty (GUM) [33]. The performance criteria for all RT-qPCR assays were computed using the formulas provided in the Supplementary Information Table S1. ...
... The performance criteria for all RT-qPCR assays were determined following the principles outlined in the guideline [33], and the calculated criteria are presented in Table 4. ...
Background
Yeast biosynthesizes fusel alcohols in fermentation through amino acid catabolism via the Ehrlich pathway. ARO8 and ARO9 genes are involved in the first step of the Ehrlich pathway, while ADH2 and ADH5 genes are involved in the last step. In this study, we describe RT-qPCR methods to determine the gene expression level of genes (ARO8, ARO9, ADH2, ADH5) found in Saccharomyces cerevisiae (Sc) and Metschnikowia pulcherrima (Mp) strains growth pasteurized white grape juice.
Methods and results
We used RNA extraction and cDNA synthesis protocols. The RT-qPCR efficiency of primer pairs was evaluated by generating a standard curve through serial dilution of yeast-derived cDNA. Method performance criteria were determined for each RT-qPCR assay. Then, we evaluated the gene expression levels of the four genes in all samples. RNA extraction and cDNA synthesis from yeast samples demonstrated the method’s capability to generate high-yield, high-purity nucleic acids, supporting further RT-qPCR analysis. The highest normalized gene expression levels of ARO8 and ARO9 were observed in SC1, SC4, and SC5 samples. No significant difference in ADH2 gene expression among Mp strains was observed during the examination of ADH2 and ADH5 genes (p < 0.05). We observed no expression of the ADH5 gene in Mp strains except MP6 strain. The expression of ADH2 and ADH5 genes was higher in Sc strains compared to Mp strains.
Conclusions
The results suggest that the proposed RT-qPCR methods can measure gene expression of ARO8, ARO9, ADH2, and ADH5 in Sc and Mp strains growing in pasteurized white grape juice.
... The measurement uncertainty was calculated from collaborative trial data according to the document 'Guidance Document on Measurement Uncertainty for GMO Testing Laboratories'. 29 For the feed sample, instead, the measurement uncertainty was calculated using data obtained on the sample according to new EURL' document 'Guidance Document on Measurement Uncertainty for GMO Testing Laboratories, third edition'. 30 Estimation of absolute and practical LOD and LOQ LOD and LOQ practical values were estimated as the ratio between the absolute limit of detection (LOD abs ) or the absolute limit of quantification (LOQ abs ) of the event-specific qPCR method associated with the sample and the measured copy number of its corresponding reference gene in the extracted DNA. ...
... As expected, for the CRMs GTS 40-3-2 and CRM MON 810 the bias % and RSDr % were within the acceptance criteria. 16,29 Even taking into account the contribution of DNA extraction, the RSDr % was similar between the four methods. ...
Background
The analysis of genetically modified organisms (GMOs) in food and feed is broadly based on the detection of target DNA sequences using PCR‐based methods. The first step of a typical workflow in a GMO testing laboratory is DNA extraction and purification. Several DNA extraction methods have been described for this purpose so far. Whatever the method adopted, the yield and the quality of the extracted DNA are essential factors for the success of GMO analysis.
Results
The DNA extraction yield was variable between the different methods, while quality and efficiency appeared comparable. Each method allowed a different yield and a slight correlation between inhibition and the food/feed matrix extracted was observed. Particularly for challenging matrices, the method used for DNA extraction has a remarkable influence on both the quality and quantity of the recovered DNA. Small variations in value % GM content were observed in complex, heterogeneous matrices but not in CRMs and homogeneous matrices.
Conclusions
In this study, the purpose is not to establish the best method of extraction but to examine several parameters that play an important role in GMO detection and verify a possible effect on the quantified GMO percentage. In particular, it was found that the yield and quality of extracted DNA depends on the type of sample, notably the degree of processing, composition, particle size, and the protocol used, especially in relation to sample intake. The slightly different GM content observed in heterogeneous matrices could raise doubt in the case of samples close to the legal threshold.
... There have been several guidelines for the validation of PCR-based methods within the scope of ISO 17025 (EURL, 2008;Trapman et al., 2009), and mostly, they were used for the validation of detection and quantification methods for genetically modified organisms. In this study, it was aimed to develop of SYBR green-based real time PCR assays for detection of adulteration in wheat-based composite breads and their in-house validation. ...
... The cereal species and their ratios in flour mixtures and the breads produced from these flours were determined by using SYBR green-based real time PCR assays. In this study, PCR assay performance criteria were evaluated and validated within the scope of ISO 17025 (EURL, 2008;Trapman et al., 2009). ...
Supplementation of wheat bread with maize, rye and oat flours provides higher amounts of protein, dietary fibres, antioxidants, minerals and vitamins. Minimum levels of non-wheat flours in composite breads are regulated by legislation. Thus, objective methods are needed to detect their supplementation levels in bread. In this study, convenient SYBR green-based real time PCR assays using genome specific primers were developed for species identification and quantification in wheat-based composite breads. Three PCR assays targeting gliadin, pML1, secalin and avenin genes for wheat, maize, rye and oat were validated by considerations of a single laboratory procedure. The cereal flour contents in bread with 20% maize, 30% rye and 15% oat flours were quantified as 21.46% ± 3.90%, 34.43% ± 7.12% and 12.54% ± 3.59%, respectively. The limit of detection and limit of quantification values of the assays were 0.086%, 0.410% for maize, 0.166%, 0.808% for rye and 0.119%, 0.290% for oat in wheat-based composite bread. These assays were able to identify and quantify cereal flours in bread successfully and have potential as rapid and sensitive methods for routine detection and quantification of adulteration in wheat-based composite breads. This approach is also applicable to the analysis of all bakery products including any variety of wheat, maize, rye, oat.
... The variability associated with the repeatability, intermediate precision and bias can be used to estimate the measurement uncertainty of a dPCR result. The general principles provided in the technical report from Trapmann et al. (2009) to estimate the analytical variability of quantitative analytical results obtained by real-time PCR can also be applied for dPCR methods. The approach detailed below to determine the uncertainty associated to a dPCR result is using data derived from within-laboratory samples (ISO 5725-2; Linsinger, 2008). ...
... In addition, the calibration curve provides a single measurement result for the laboratory with close threshold cycle (Ct) values. It is also essential to insert an internal quality control (analytical standard from reference material) to ensure that the performance of the analytical process remains effectively unchanged (Trapman et al., 2009). ...
... The variability associated with the repeatability, intermediate precision and bias can be used to estimate the measurement uncertainty of a dPCR result. The general principles provided in the technical report from Trapmann et al. (2009) to estimate the analytical variability of quantitative analytical results obtained by real-time PCR can also be applied for dPCR methods. The approach detailed below to determine the uncertainty associated to a dPCR result is using data derived from within-laboratory samples (ISO 5725-2; Linsinger, 2008). ...
The so-called digital Polymerase Chain Reaction (dPCR) is a relatively new technique for the detection and quantification of DNA, but its application in analytical laboratories is steadily increasing. In contrast to quantitative real-time PCR, DNA (fragments) can be quantified here without the need for calibration curves. Using dPCR, the PCR mix containing the (target) DNA is partitioned – depending on the device used – currently into a maximum of 10,000,000 small compartments with a volume as low as a few picolitres. These can be either physically distinct compartments on a chip (referred to as chamber-based digital PCR [cdPCR]), or the compartments correspond to water-in-oil droplets (referred to as droplet digital [ddPCR]). Once the PCR has been carried out simultaneously in all compartments/droplets, it is common to both approaches that the number of positive and negative signals for each partition is counted by a fluorescence measurement.
With this technique, an absolute quantification of DNA copy numbers can be performed with high precision and trueness, even for very low DNA copy numbers. Furthermore, dPCR is considered less susceptible than qPCR to PCR inhibitory substances that could be co-extracted during DNA extraction from different samples.
Digital PCR has already been applied in various fields, for example for the detection and quantification of GMOs, species (animals, plants), human disease bioindicators, food viruses and bacteria including pathogens.
When establishing dPCR in a laboratory, different aspects have to be considered. These include, but are not limited to, the adjustment of the type of the PCR master mix used, optimised primer and probe concentrations and the signal separation of positive and negative compartments. This document addresses these and other aspects and provides recommendations for the transfer of existing real-time PCR methods into a dPCR format.
... based on endogenous genes or proteins and these might differ in landraces and wild relatives. Existing guidelines outlining the technical issues related to the estimation of measurement uncertainty (MU) do consider parameters associated with the dispersion of the values(Trapmann et al., 2009). However, once again these are relative to the MU associated with an analytical result only when deciding whether that result falls within the legislation for food and feed control purposes.Detection methods also have intrinsic problems that become particularly relevant when working with landraces or wild relatives. ...
The flow of transgenes into landraces and wild relatives is an important biosafety concern. The case of transgene flow into local maize varieties in Mexico (the center of origin of maize) has been intensively debated over the past 15 years, including legal, political, and environmental disputes fanned by the existence of a significant scientific controversy over the methods used for the detection of transgenes. The use of diverse approaches and a lack of harmonized methods specific to the detection and monitoring of transgenes in landraces have generated both positive and negative results regarding contamination of Mexican maize with genetically modified material over the years. In this paper, we revisit the case of transgene contamination in Mexican maize and present a novel research approach based on socio-biological analysis of contrasting communities and seed management systems. Two communities were used to investigate how different social and biological factors can affect transgene flow and impact transgene spread in Mexico. Our results show the presence of transgenes in one community and thus support the position that transgenes are highly likely to be present in Mexican maize landraces. However, our work also demonstrates that the extent and frequency with which transgenes can be found will significantly depend on the societal characteristics and seed management systems of the local communities. Therefore, we argue that future analysis of transgene presence should include social research on the seed management practices in the sampling area so that more robust and comprehensive understandings and conclusions can be drawn.
... Für eine der am weitesten verbreiteten molekularbiologischen Methoden, die Echtzeit-Polymerase-Kettenreaktion (real time Polymerase Chain Reaction, rt-PCR), wurde eine Anleitung, wie die Messunsicherheit zu ermitteln ist, mit Beispielen aus der Quantifizierung genetisch modifizierter DNA publiziert. 76) Eine Entwicklung in der fundamentalen Metrologie wird von Chemikern bisher kaum wahrgenommen: Die Metrologie-Institute, die im Internationalen Komitee für Maße und Gewichte (Comité International des Poids et Mesures, CIPM) zusammenarbeiten, versuchen, zukünftig die Basiseinheiten des internationalen Maßsystems (SI) über exakt festgelegte Naturkonstanten zu definieren. 77 ...
... No Guidance document on measurement uncertainty for GMO testing laboratories do JRC são estabelecidas formas de estimar a incerteza da medição para métodos quantitativos de análises de OGM por PCR em Tempo Real. No referido documento é citado ainda que, apesar do reconhecimento da importância da estimativa da incerteza de medição em análises qualitativas, os trabalhos sobre esta questão ainda estão em fase de desenvolvimento 38 . ...
A informacao sobre a origem transgenica de alimentos e muito relevante. Conforme a legislacao brasileira e de outros paises, o consumidor deve ser informado da natureza transgenica dos alimentos ou ingredientes que contenham ou que sejam produzidos a partir de organismos geneticamente modificados (OGM), com presenca acima de um limite estabelecido. A necessidade de monitorar a presenca e determinar o percentual de OGM em alimentos tem gerado uma constante demanda pelo desenvolvimento de metodologias capazes de detectar, identificar e quantificar o DNA exogeno. Entretanto, esses metodos necessitam ser validados para garantir confiabilidade aos resultados. No presente trabalho, a validacao de metodos para deteccao de soja Roundup Ready® em graos e produtos de soja por reacao em cadeia de polimerase foi contextualizada.Considerando-se atuais tendencias em validacao de metodos, os guias para validacao de metodos especificos para OGM nao contemplam todos os parâmetros necessarios para avaliar a adequacao aos propositos de uso dos metodos, principalmente no caso de metodos qualitativos. Os parâmetros de desempenho mais frequentemente citados na literatura foram precisao (repetitividade), sensibilidade, linearidade e veracidade para metodologias quantitativas e taxas de sensibilidade e seletividade para qualitativas. Contudo, importantes parâmetros tem sido negligenciados nos processos de validacao de metodos deste escopo analitico. (AU) Information on the origin of transgenic foods is quite relevant. According to the Brazilian and othercountries legislations, the consumer must be informed about the nature of transgenic foods andingredients, which contain or being produced from genetically modified organisms (GMOs), in amountabove the established limit. Thus, there is a growing need for evaluating the occurrence of GMOs, and todetermine the percentage of them in food. This need has generated a demand for developing methodsto detect, identify and quantify the exogenous DNA. However, these methods need to be validated toensure reliability of the results. This review paper evaluated the validation of methodologies for detectingthe Roundup Ready® soybean in grain and soybean products by polymerase chain reaction. Consideringthe current trends in method validation, the specific guides for validating GMOs do not include all ofthe parameters required to assess the fitness for its purpose, especially in the case of qualitative methods.The most frequent parameters reported in the literature were accuracy, precision (repeatability), linearityand sensitivity for quantitative methods, and rates of sensitivity and selectivity for qualitative methods.However, important parameters have been neglected in the validation procedures of this analytical scope.(AU)
... This holds also true for quantification of GMO at all levels ? not only at 0.1% [8]. Quantification of GMO adds further obstacles to the measuring procedure: After quantitative real-time PCR of both the transgene and a species-specific reference gene, the corresponding mass fraction has to be calculated considering the (assumed) zygosity of the plant tissue(s) and plant species under investigation [9]. ...
HINT:
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Background
According to Regulation (EU) No 619/2011, trace amounts of non-authorised genetically modified organisms (GMO) in feed are tolerated within the EU if certain prerequisites are met. Tolerable traces must not exceed the so-called `minimum required performance limit? (MRPL), which was defined according to the mentioned regulation to correspond to 0.1% mass fraction per ingredient. Therefore, not yet authorised GMO (and some GMO whose approvals have expired) have to be quantified at very low level following the qualitative detection in genomic DNA extracted from feed samples. As the results of quantitative analysis can imply severe legal and financial consequences for producers or distributors of feed, the quantification results need to be utterly reliable.
Results
We developed a statistical approach to investigate the experimental measurement variability within one 96-well PCR plate. This approach visualises the frequency distribution as zygosity-corrected relative content of genetically modified material resulting from different combinations of transgene and reference gene Cq values. One application of it is the simulation of the consequences of varying parameters on measurement results. Parameters could be for example replicate numbers or baseline and threshold settings, measurement results could be for example median (class) and relative standard deviation (RSD). All calculations can be done using the built-in functions of Excel without any need for programming. The developed Excel spreadsheets are available (see section `Availability of supporting data? for details). In most cases, the combination of four PCR replicates for each of the two DNA isolations already resulted in a relative standard deviation of 15% or less.
Conclusions
The aims of the study are scientifically based suggestions for minimisation of uncertainty of measurement especially in ?but not limited to? the field of GMO quantification at low concentration levels. Four PCR replicates for each of the two DNA isolations seem to be a reasonable minimum number to narrow down the possible spread of results.
... In the case of microbiological laboratories performing molecular testing for the detection and quantification of genetically modified organisms (GMOs), measurement uncertainty is estimated according to JRC/IRMM Guidance EUR 22756 EN [19]. • disposable equipment should be clean and sterile when appropriate; • re-used glassware should be properly cleaned and sterilised when appropriate; • ideally, laboratories should have a separate autoclave for decontamination. ...
This document provides microbiological laboratories with appropriate information and guidance on how to fulfill the requirements of ISO/IEC 17025 on Accreditation of Analytical Laboratories
... They mainly aim at quantifying the uncertainty [9], providing a frame to set up single-laboratory validations of methods of analysis [10] and describing the relationship between analytical results and measurement uncertainty. In such documents, particular focus is placed on the provisions of EU legislation concerning contaminants in food and undesirable substances in feed [11]. In the field of GM food and feed, a practical guidance to laboratories was published in the context of the activities of the European Network of GMO Laboratories [12] where, in particular, the concept of measurement uncertainty was detailed. ...
Key to sound validation studies is the formalization and harmonization of procedures for design of experiment and interpretation
of results. International guidelines (ISO 5725, ENGL) are available for the validation of GMO detection methods, and ad-hoc validation statistics (e.g. per cent bias, repeatability and reproducibility) are used for in-house and inter-laboratory
testing and decision-making. Acceptability criteria have been set but not every situation can be covered by a preset rule;
the interpretation of results in validation largely depends on expert judgement being a matter of professional judgment and
expertise. Fuzzy logic-based techniques may be used to summarize the information obtained by independent validation statistics
and are helpful in such respect. A comprehensive indicator of method performance permits direct comparison between methods
and facilitates the evaluation of multiple, yet contradictory statistics. The European Union Reference Laboratory for GM Food
and Feed has already proposed the fuzzy principle in the context of method validation. Other studies have also proved its
applicability in other areas of GMO analysis, but the application has been limited hitherto. In this article, we review the
fuzzy logic principle and its potential to support the continuous progress of GMO science and routine laboratory analyses.
KeywordsFuzzy logic-Genetically modified organisms-Real time quantitative polymerase chain reaction (qPCR)-Validation of methods
... The LOD can be defined as the minimum amount or concentration of the analyte in a test sample that can be detected reliably but not necessarily quantified, as demonstrated by a collaborative trial or other appropriate validation, whereas the LOQ is the lowest concentration or amount of the analyte in a test sample that can be quantitatively determined with an acceptable level of precision and accuracy, as demonstrated by a collaborative trial or other appropriate validation (24). Looking at the literature, for real-time PCR methods, we find numerous different approaches proposed and/or adopted for this purpose (25)(26)(27)(28)(29)(30)(31)(32)(33)(34). Among all of these approaches, the most widely adopted consists of pointing out the lowest amount or concentration of the analyte, where it can be detected at least 95% of the time, ensuring e5% false negative results. ...
The comparison of five real-time polymerase chain reaction (PCR) methods targeted at maize ( Zea mays ) endogenous sequences is reported. PCR targets were the alcohol dehydrogenase (adh) gene for three methods and high-mobility group (hmg) gene for the other two. The five real-time PCR methods have been checked under repeatability conditions at several dilution levels on both pooled DNA template from several genetically modified (GM) maize certified reference materials (CRMs) and single CRM DNA extracts. Slopes and R(2) coefficients of all of the curves obtained from the adopted regression model were compared within the same method and among all of the five methods, and the limit of detection and limit of quantitation were analyzed for each PCR system. Furthermore, method equivalency was evaluated on the basis of the ability to estimate the target haploid genome copy number at each concentration level. Results indicated that, among the five methods tested, one of the hmg-targeted PCR systems can be considered equivalent to the others but shows the best regression parameters and a higher repeteability along the dilution range. Thereby, it is proposed as a valid module to be coupled to different event-specific real-time PCR for maize genetically modified organism (GMO) quantitation. The resulting practicability improvement on the analytical control of GMOs is discussed.
Measurement uncertainty (MU) based on the top-down approach is crucial for the diagnostic laboratory following the WOAH guideline. Real-time RT-PCR is one of the laboratory methods for detection of influenza A virus subtype H5 in chicken samples used by the National Institute of Animal Health (NIAH). The MU of testing provides a confident result and complies with the ISO/IEC 17025 requirements. The objective is to estimate the MU of real-time RT-PCR for the detection of influenza A virus subtype H5 in different chicken matrices using a top-down approach. Influenza virus subtype H5 RNA was diluted in AVE buffer and three different sample matrices: oropharyngeal swab, lung, and chicken meat. Each sample was prepared from ten samples. The cutoff Ct values of real-time RT-PCR in different matrices were determined, and one-way ANOVA was used to compare these cutoff values. Weak positive RNA was collected for evaluating the measurement uncertainty of real-time RT-PCR for detection of Influenza A virus subtype H5 in different chicken matrices using a top-down approach. The cutoff values of the oropharyngeal swab, lung, and chicken meat were 35, 33, and 36 cycles at the RNA concentration of 1×10 1 , 1×10 2 และ 1×10 2 copies/reaction, respectively. There were statistically significant differences (p<0.05). The expanded uncertainty was 0.0486. And the MU (95% CI) of real-time RT-PCR for detection of influenza A virus subtype H5 in oropharyngeal swab, lung, and chicken meat matrices were 33.25-36.75, 31.25-34.75, and 37.25-40.75 cycles, respectively. The difference of sample matrices affected the cutoff and the MU range of the real-time RT-PCR technique for detecting influenza A virus subtype H5. The top-down approach can be applied for internal quality control in a veterinary diagnostic laboratory or used as a management guideline for sample collection.
The transgenic rice G6H1 was a new event with the traits of herbicide-tolerance and insect-resistant. Herein, we developed one event-specific real-time PCR method with high specificity and sensitivity for G6H1 event quantitative analysis, and validated its performance on practical samples quantification through a collaborative ring trial. A total of eight laboratories participated in this validation and quantified three blind G6H1 powder samples including DNA extraction and real-time PCR analysis. The statistical analyzed results from returned data confirmed its high PCR efficiency, and good linearity, trueness and precision, indicating that the developed G6H1 real-time PCR assay were accurate, reliable, and comparable for G6H1 identification and quantification.
Bringing together the ideas of experts from around the world, this incisive text offers cutting-edge perspectives on the risk analysis and governance of genetically modified organisms (GMOs), supporting effective and informed decision-making in developing countries. Comprised of four comprehensive sections, this book covers: integrated risk analysis and decision making, giving an overview of the science involved and examining risk analysis methods that impact decision-making on the release of GMOs, particularly in developing countries; diversification of expertise involved in risk analysis and practical ways in which the lack of expertise in developing countries can be overcome; risk analysis based regulatory systems and how they can be undermined by power relationships and socio-political interests, as well as strategies for improving GMO policy development and regulatory decision-making; and case studies from developing countries providing lessons based on real-world experience that can inform our current thinking.
The annual report of the JRC Institute for Reference Materials and Measurements (IRMM) describes the research highlights in 2009.
The presence of GMOs in food and feed is mainly detected by the use of targets focusing on promoters and terminators. As some genes are frequently used in GM construction, they also constitute excellent screening elements and their use is increasing. In this work, we propose a new target for the detection of cry1Ab and cry1Ac genes by real-time PCR and pyrosequencing. The specificity, sensitivity and robustness of the real-time PCR method were tested following the recommendations of international guidelines and the method met the expected performance criteria. This paper also shows how the robustness testing was assessed. This new cry1Ab/Ac method is able to provide a positive signal with a larger number of GM events than the other existing methods using double dye-probes. The method permits to analyse the results with less ambiguity than the SYBRGreen method recommended by EU-RL GMFF. A pyrosequencing method was also developed to gain additional information thanks to the sequence of the amplicon. This method of sequencing-by-synthesis is able to determine the sequence between the primers used for PCR. The pyrosequencing showed that the sequences internal to the primers present differences following the GM events considered and three different sequences were observed. The sensitivity of the pyrosequencing was tested on reference flours at low GM percentage and different copy numbers. Improvements of the pyrosequencing protocol provides correct sequences with 50 copies of the target. Below this copy number, the quality of the sequence was more random.
The present technical report deals with monitoring the
efficiency of measures/strategies for coexistence between
genetically modified (GM) and non-GM maize crop production.
The report is a follow up of the best practices for coexistence
in maize crop production proposed by the Technical Working
Group (TWG) for Maize of the European Coexistence Bureau
(ECoB).
The ECoB TWG Maize held three meetings in October 2010,
June 2012 and November 2012 and examined state-ofart-
knowledge from scientific literature, research projects
and empirical evidence provided by numerous finished and
ongoing studies looking at the appropriate level of monitoring,
monitoring strategy, sampling and testing issues, detection
methods, analysis of results and possible follow up.
The review of this information (coming from a total of 55
references) is presented in a structured manner in Section 3
and 4 of the document.
The overview of the activities carried out by EU Member
States for monitoring effectiveness/efficiency of coexistence
measures in maize crop production (Section 3), shows a
still limited experience in practical terms, due to the limited
experience in commercial cultivation of GM maize in most
EU Member States. However, the present report provides
technical guidance to those responsible for monitoring the
efficiency of coexistence strategies.
Following a report on a significant amount of horse DNA being detected in a beef burger product on sale to the public at a UK supermarket in early 2013, the Elliott report was published in 2014 and contained a list of recommendations for helping ensure food integrity. One of the recommendations included improving laboratory testing capacity and capability to ensure a harmonised approach for testing for food authenticity. Molecular biologists have developed exquisitely sensitive methods based on the polymerase chain reaction (PCR) or mass spectrometry for detecting the presence of particular nucleic acid or peptide/protein sequences. These methods have been shown to be specific and sensitive in terms of lower limits of applicability, but they are largely qualitative in nature. Historically, the conversion of these qualitative techniques into reliable quantitative methods has been beset with problems even when used on relatively simple sample matrices. When the methods are applied to complex sample matrices, as found in many foods, the problems are magnified resulting in a high measurement uncertainty associated with the result which may mean that the assay is not fit for purpose. However, recent advances in the technology and the understanding of molecular biology approaches have further given rise to the re-assessment of these methods for their quantitative potential. This review focuses on important issues for consideration when validating a molecular biology assay and the various factors that can impact on the measurement uncertainty of a result associated with molecular biology approaches used in detection of food fraud, with a particular focus on quantitative PCR-based and proteomics assays.
For transferring the event-specific PCR methods of genetically modified papaya Huanong No.1 to other laboratories, we validated the previous developed PCR assays of Huanong No.1 according to the international standard organization (ISO) guidelines. A total of 11 laboratories participated and returned their test results in this trial. In qualitative PCR assay, the high specificity and limit of detection as low as 0.1% was confirmed. For the quantitative PCR assay, the limit of quantification was as low as 25 copies. The quantitative biases among ten blind samples were within the range between 0.21% and 10.04%. Furthermore, the measurement uncertainty of the quantitative PCR results was calculated within the range between 0.28% and 2.92% for these ten samples. All results demonstrated that the Huanong No.1 qualitative and quantitative PCR assays were creditable and applicable for identification and quantification of GM papaya Huanong No.1 in further routine lab analysis.
The Cauliflower mosaic virus (CaMV) 35S promoter (P35S) is a commonly used target for detection of genetically modified organisms (GMOs). There are currently 24 reported detection methods, targeting different regions of the P35S promoter. Initial assessment revealed that due to the absence of primer binding sites in the P35S sequence, 19 of the 24 reported methods failed to detect P35S in MON88913 cotton, and the other two methods could only be applied to certain GMOs. The rest three reported methods were not suitable for measurement of P35S in some testing events, because SNPs in binding sites of the primer/probe would result in abnormal amplification plots and poor linear regression parameters. In this study, we discovered a conserved region in the P35S sequence through sequencing of P35S promoters from multiple transgenic events, and developed new qualitative and quantitative detection systems targeting this conserved region. The qualitative PCR could detect the P35S promoter in 23 unique GMO events with high specificity and sensitivity. The quantitative method was suitable for measurement of P35S promoter, exhibiting good agreement between the amount of template and Ct values for each testing event. This study provides a general P35S screening method, with greater coverage than existing methods.
Cultivation and marketing of genetically modified organisms (GMOs) have been unevenly adopted worldwide. To facilitate international trade and to provide information to consumers, labelling requirements have been set up in many countries. Quantitative real-time polymerase chain reaction (qPCR) is currently the method of choice for detection, identification and quantification of GMOs. This has been critically assessed and the requirements for the method performance have been set. Nevertheless, there are challenges that should still be highlighted, such as measuring the quantity and quality of DNA, and determining the qPCR efficiency, possible sequence mismatches, characteristics of taxon-specific genes and appropriate units of measurement, as these remain potential sources of measurement uncertainty. To overcome these problems and to cope with the continuous increase in the number and variety of GMOs, new approaches are needed. Statistical strategies of quantification have already been proposed and expanded with the development of digital PCR. The first attempts have been made to use new generation sequencing also for quantitative purposes, although accurate quantification of the contents of GMOs using this technology is still a challenge for the future, and especially for mixed samples. New approaches are needed also for the quantification of stacks, and for potential quantification of organisms produced by new plant breeding techniques.
Die analytische Chemie nutzt vermehrt optoelektronische Techniken mit mikrostrukturierten Komponenten. Nach Chemosensoren beherrschen nun Biosensoren das Feld. Ausschließlich chemisch synthetisierte Rezeptoren bleiben gegenüber molekularbiologischen Reaktionspartnern zurück. In Diagnostik und Lebensmittelkontrolle ist die Zeit autonomer Analysenplattformen angebrochen. Auffällig ist die Dominanz massenspektrometrischer Techniken, sowohl in Kopplung, als auch als Stand-alone-Verfahren. Generell geht der Trend in Richtung Life-Science-Anwendungen, während die Umweltanalytik fast vollständig verschwindet.
In this study, a collaborative trial of validating a real-time PCR method for TT51-1 rice event was organized including six participating laboratories. In this validation, serially diluted solutions from homogenous genomic DNA of TT51-1 event were used to construct standard curves of TT51-1 event and Phospholipase D (PLD) assays. The PCR efficiency was 95%, and the R2 coefficient was 0.99 for the TT51-1 system. The mean quantitative values for blind samples containing 0.1%, 0.5% 1%, 5%, and 10% (w/w) TT51-1, corresponded to 0.1%, 0.51%, 1.06%, 4.83%, and 9.62%, respectively, with a bias (%) ranging from -3.77% to 5.87%. The repeatability and reproducibility were all below 25% across the entire dynamic range. Furthermore, the measurement uncertainties of the quantitative results were estimated to be 0.10%, 0.20%, 0.40%, 1.76%, 3.52% (w/w) for the tested samples. Both the LOD and LOQ were calculated to be 0.22%. This collaborative trial demonstrated that the TT51-1 method produces reliable, comparable, and reproducible results for a given sample set, and can be adopted as a detection standard for testing laboratories.
Randomised experimental designs are considered statistically advantageous for many scientific applications, as they reduce
the effect of problematic parameters such as sample position. The effect of randomisation was assessed using real-time PCR
experiments for the determination of genetically modified material. Replicate microtitre plates using a randomised design
were compared with replicate microtitre plates that contained the same samples allocated in a systematic fashion to experimental
position within the plate. Results indicated that randomisation would help reduce between-plate variability and would give
advantages in intra-laboratory studies. However, these benefits may be less effective for inter-laboratory studies as the
between-laboratory variability often far exceeds the between-plate variability.
KeywordsRandomisation–Intra-laboratory–Inter-laboratory–Fitness for purpose–Real-time quantitative PCR–GM analysis
Genetically modified plants, in the following referred to as genetically modified organisms or GMOs, have been commercially grown for almost two decades. In 2010 approximately 10% of the total global crop acreage was planted with GMOs (James, 2011). More than 30 countries have been growing commercial GMOs, and many more have performed field trials. Although the majority of commercial GMOs both in terms of acreage and specific events belong to the four species: soybean, maize, cotton and rapeseed, there are another 20+ species where GMOs are commercialized or in the pipeline for commercialization. The number of GMOs cultivated in field trials or for commercial production has constantly increased during this time period. So have the number of species, the number of countries involved, the diversity of novel (added) genetic elements and the global trade. All of these factors contribute to the increasing complexity of detecting and correctly identifying GMO derived material. Many jurisdictions, including the European Union (EU), legally distinguish between authorized (and therefore legal) and un-authorized (and therefore illegal) GMOs. Information about the developments, field trials, authorizations, cultivation, trade and observations made in the official GMO control laboratories in different countries around the world is often limited, despite several attempts such as the OECD BioTrack for voluntary dissemination of data. This lack of information inevitably makes it challenging to detect and identify GMOs, especially the un-authorized GMOs. The present paper reviews the state of the art technologies and approaches in light of coverage, practicability, sensitivity and limitations. Emphasis is put on exemplifying practical detection of un-authorized GMOs. Although this paper has a European (EU) bias when examples are given, the contents have global relevance.
Statistics -Vocabulary and symbols -Part 1: General statistical terms and terms used in probability
- Iso Fdis
ISO/FDIS 3534-1 (2006): Statistics -Vocabulary and symbols -Part 1: General statistical terms and terms
used in probability
Proposed draft guidelines for evaluating acceptable methods of analysis [23] IUPAC recommendation (1995): Nomenclature in evaluation of analytical methods including detection and quantification capabilities
- Cx Codex
- Mas
Codex CX/MAS 02/4 (2002): Proposed draft guidelines for evaluating acceptable methods of analysis
[23] IUPAC recommendation (1995): Nomenclature in evaluation of analytical methods including detection and
quantification capabilities, Pure & Appl. Chem., 67(10), 1699-1723
The Certification of a new set of Reference Materials of Soya Powder with different Mass Fractions of Roundup Ready TM Soya
- S Trapmann
- Le Guern
- L Kramer
- Gn Schimmel
- H Pauwels
- J Anklam
- E Van Den Eede
- G Brodmann
Trapmann S, Le Guern L, Kramer GN, Schimmel H, Pauwels J, Anklam E, Van den Eede G, Brodmann P
(2000): The Certification of a new set of Reference Materials of Soya Powder with different Mass Fractions of
Roundup Ready
TM Soya, EC certification report EUR 19573 EN, ISBN 92-828-9639-0