共查询到20条相似文献,搜索用时 46 毫秒
1.
Validation of a newly developed hexaplex real-time PCR assay for screening for presence of GMOs in food, feed and seed 总被引:1,自引:0,他引:1
C. Bahrdt A. B. Krech A. Wurz D. Wulff 《Analytical and bioanalytical chemistry》2010,396(6):2103-2112
For years, an increasing number and diversity of genetically modified plants has been grown on a commercial scale. The need
for detection and identification of these genetically modified organisms (GMOs) calls for broad and at the same time flexible
high throughput testing methods. Here we describe the development and validation of a hexaplex real-time polymerase chain
reaction (PCR) screening assay covering more than 100 approved GMOs containing at least one of the GMO targets of the assay.
The assay comprises detection systems for Cauliflower Mosaic Virus 35S promoter, Agrobacterium tumefaciens NOS terminator, Figwort Mosaic Virus 34S promoter and two construct-specific sequences present in novel genetically modified
soybean and maize that lack common screening elements. Additionally a detection system for an internal positive control (IPC)
indicating the presence or absence of PCR inhibiting substances was included. The six real-time PCR systems were allocated
to five detection channels showing no significant crosstalk between the detection channels. As part of an extensive validation,
a limit of detection (LODabs) ≤ ten target copies was proven in hexaplex format. A sensitivity ≤ ten target copies of each GMO detection system was still
shown in highly asymmetric target situations in the presence of 1,000 copies of all other GMO targets of each detection channel.
Furthermore, the applicability to a broad sample spectrum and reliable indication of inhibition by the IPC system was demonstrated.
The presented hexaplex assay offers sensitive and reliable detection of GMOs in processed and unprocessed food, feed and seed
samples with high efficiency. 相似文献
2.
See what you eat—broad GMO screening with microarrays 总被引:1,自引:0,他引:1
Franz von Götz 《Analytical and bioanalytical chemistry》2010,396(6):1961-1967
Despite the controversy of whether genetically modified organisms (GMOs) are beneficial or harmful for humans, animals, and/or ecosystems, the number of cultivated GMOs is increasing every year. Many countries and federations have implemented safety and surveillance systems for GMOs. Potent testing technologies need to be developed and implemented to monitor the increasing number of GMOs. First, these GMO tests need to be comprehensive, i.e., should detect all, or at least the most important, GMOs on the market. This type of GMO screening requires a high degree of parallel tests or multiplexing. To date, DNA microarrays have the highest number of multiplexing capabilities when nucleic acids are analyzed. This trend article focuses on the evolution of DNA microarrays for GMO testing. Over the last 7 years, combinations of multiplex PCR detection and microarray detection have been developed to qualitatively assess the presence of GMOs. One example is the commercially available DualChip® GMO (Eppendorf, Germany; http://www.eppendorf-biochip.com), which is the only GMO screening system successfully validated in a multicenter study. With use of innovative amplification techniques, promising steps have recently been taken to make GMO detection with microarrays quantitative. Figure
EU-validated GMO screening microarray 相似文献
3.
Advances in molecular techniques for the detection and quantification of genetically modified organisms 总被引:3,自引:1,他引:2
Elenis DS Kalogianni DP Glynou K Ioannou PC Christopoulos TK 《Analytical and bioanalytical chemistry》2008,392(3):347-354
Progress in genetic engineering has led to the introduction of genetically modified organisms (GMOs) whose genomes have been
altered by the integration of a novel sequence conferring a new trait. To allow consumers an informed choice, many countries
require food products to be labeled if the GMO content exceeds a certain threshold. Consequently, the development of analytical
methods for GMO screening and quantification is of great interest. Exponential amplification by the polymerase chain reaction
(PCR) remains a central step in molecular methods of GMO detection and quantification. In order to meet the challenge posed
by the continuously increasing number of GMOs, various multiplex assays have been developed for the simultaneous amplification
and/or detection of several GMOs. Classical agarose gel electrophoresis is being replaced by capillary electrophoresis (CE)
systems, including CE chips, for the rapid and automatable separation of amplified fragments. Microtiter well-based hybridization
assays allow high-throughput analysis of many samples in a single plate. Microarrays have been introduced in GMO screening
as a technique for the simultaneous multianalyte detection of amplified sequences. Various types of biosensors, including
surface plasmon resonance sensors, quartz crystal microbalance piezoelectric sensors, thin-film optical sensors, dry-reagent
dipstick-type sensors and electrochemical sensors were introduced in GMO screening because they offer simplicity and lower
cost. GMO quantification is performed by real-time PCR (rt-QPCR) and competitive PCR. New endogenous reference genes have
been validated. rt-QPCR is the most widely used approach. Multiplexing is another trend in this field. Strategies for high-throughput
multiplex competitive quantitative PCR have been reported. 相似文献
4.
Validation of PCR methods for quantitation of genetically modified plants in food. 总被引:19,自引:0,他引:19
For enforcement of the recently introduced labeling threshold for genetically modified organisms (GMOs) in food ingredients, quantitative detection methods such as quantitative competitive (QC-PCR) and real-time PCR are applied by official food control laboratories. The experiences of 3 European food control laboratories in validating such methods were compared to describe realistic performance characteristics of quantitative PCR detection methods. The limit of quantitation (LOQ) of GMO-specific, real-time PCR was experimentally determined to reach 30-50 target molecules, which is close to theoretical prediction. Starting PCR with 200 ng genomic plant DNA, the LOQ depends primarily on the genome size of the target plant and ranges from 0.02% for rice to 0.7% for wheat. The precision of quantitative PCR detection methods, expressed as relative standard deviation (RSD), varied from 10 to 30%. Using Bt176 corn containing test samples and applying Bt176 specific QC-PCR, mean values deviated from true values by -7to 18%, with an average of 2+/-10%. Ruggedness of real-time PCR detection methods was assessed in an interlaboratory study analyzing commercial, homogeneous food samples. Roundup Ready soybean DNA contents were determined in the range of 0.3 to 36%, relative to soybean DNA, with RSDs of about 25%. Taking the precision of quantitative PCR detection methods into account, suitable sample plans and sample sizes for GMO analysis are suggested. Because quantitative GMO detection methods measure GMO contents of samples in relation to reference material (calibrants), high priority must be given to international agreements and standardization on certified reference materials. 相似文献
5.
New trends in bioanalytical tools for the detection of genetically modified organisms: an update 总被引:3,自引:1,他引:2
Michelini E Simoni P Cevenini L Mezzanotte L Roda A 《Analytical and bioanalytical chemistry》2008,392(3):355-367
Despite the controversies surrounding genetically modified organisms (GMOs), the production of GM crops is increasing, especially
in developing countries. Thanks to new technologies involving genetic engineering and unprecedented access to genomic resources,
the next decade will certainly see exponential growth in GMO production. Indeed, EU regulations based on the precautionary
principle require any food containing more than 0.9% GM content to be labeled as such. The implementation of these regulations
necessitates sampling protocols, the availability of certified reference materials and analytical methodologies that allow
the accurate determination of the content of GMOs. In order to qualify for the validation process, a method should fulfil
some criteria, defined as “acceptance criteria” by the European Network of GMO Laboratories (ENGL). Several methods have recently
been developed for GMO detection and quantitation, mostly based on polymerase chain reaction (PCR) technology. PCR (including
its different formats, e.g., double competitive PCR and real-time PCR) remains the technique of choice, thanks to its ability
to detect even small amounts of transgenes in raw materials and processed foods. Other approaches relying on DNA detection
are based on quartz crystal microbalance piezoelectric biosensors, dry reagent dipstick-type sensors and surface plasmon resonance
sensors. The application of visible/near-infrared (vis/NIR) spectroscopy or mass spectrometry combined with chemometrics techniques
has also been envisaged as a powerful GMO detection tool. Furthermore, in order to cope with the multiplicity of GMOs released
onto the market, the new challenge is the development of routine detection systems for the simultaneous detection of numerous
GMOs, including unknown GMOs. 相似文献
6.
Development of a qualitative, multiplex real-time PCR kit for screening of genetically modified organisms (GMOs) 总被引:3,自引:0,他引:3
Hans-Henno Dörries Ivonne Remus Astrid Grönewald Cordt Grönewald Kornelia Berghof-Jäger 《Analytical and bioanalytical chemistry》2010,396(6):2043-2054
The number of commercially available genetically modified organisms (GMOs) and therefore the diversity of possible target
sequences for molecular detection techniques are constantly increasing. As a result, GMO laboratories and the food production
industry currently are forced to apply many different methods to reliably test raw material and complex processed food products.
Screening methods have become more and more relevant to minimize the analytical effort and to make a preselection for further
analysis (e.g., specific identification or quantification of the GMO). A multiplex real-time PCR kit was developed to detect
the 35S promoter of the cauliflower mosaic virus, the terminator of the nopaline synthase gene of Agrobacterium tumefaciens, the 35S promoter from the figwort mosaic virus, and the bar gene of the soil bacterium Streptomyces hygroscopicus as the most widely used sequences in GMOs. The kit contains a second assay for the detection of plant-derived DNA to control
the quality of the often processed and refined sample material. Additionally, the plant-specific assay comprises a homologous
internal amplification control for inhibition control. The determined limits of detection for the five assays were 10 target
copies/reaction. No amplification products were observed with DNAs of 26 bacterial species, 25 yeasts, 13 molds, and 41 not
genetically modified plants. The specificity of the assays was further demonstrated to be 100% by the specific amplification
of DNA derived from reference material from 22 genetically modified crops. The applicability of the kit in routine laboratory
use was verified by testing of 50 spiked and unspiked food products. The herein described kit represents a simple and sensitive
GMO screening method for the reliable detection of multiple GMO-specific target sequences in a multiplex real-time PCR reaction. 相似文献
7.
Geoffrey Cottenet Carine Blancpain Véronique Sonnard Poh Fong Chuah 《Analytical and bioanalytical chemistry》2013,405(21):6831-6844
Considering the increase of the total cultivated land area dedicated to genetically modified organisms (GMO), the consumers’ perception toward GMO and the need to comply with various local GMO legislations, efficient and accurate analytical methods are needed for their detection and identification. Considered as the gold standard for GMO analysis, the real-time polymerase chain reaction (RTi-PCR) technology was optimised to produce a high-throughput GMO screening method. Based on simultaneous 24 multiplex RTi-PCR running on a ready-to-use 384-well plate, this new procedure allows the detection and identification of 47 targets on seven samples in duplicate. To comply with GMO analytical quality requirements, a negative and a positive control were analysed in parallel. In addition, an internal positive control was also included in each reaction well for the detection of potential PCR inhibition. Tested on non-GM materials, on different GM events and on proficiency test samples, the method offered high specificity and sensitivity with an absolute limit of detection between 1 and 16 copies depending on the target. Easy to use, fast and cost efficient, this multiplex approach fits the purpose of GMO testing laboratories. Figure
A broad multiplex real‐time PCR method for the detection and identification of GMOs 相似文献
8.
Katarzyna Grelewska-Nowotko Magdalena Żurawska-Zajfert Ewelina Żmijewska Sławomir Sowa 《Applied biochemistry and biotechnology》2018,185(1):207-220
In recent years, digital polymerase chain reaction (dPCR), a new molecular biology technique, has been gaining in popularity. Among many other applications, this technique can also be used for the detection and quantification of genetically modified organisms (GMOs) in food and feed. It might replace the currently widely used real-time PCR method (qPCR), by overcoming problems related to the PCR inhibition and the requirement of certified reference materials to be used as a calibrant. In theory, validated qPCR methods can be easily transferred to the dPCR platform. However, optimization of the PCR conditions might be necessary. In this study, we report the transfer of two validated qPCR methods for quantification of maize DAS1507 and NK603 events to the droplet dPCR (ddPCR) platform. After some optimization, both methods have been verified according to the guidance of the European Network of GMO Laboratories (ENGL) on analytical method verification (ENGL working group on “Method Verification.” (2011) Verification of Analytical Methods for GMO Testing When Implementing Interlaboratory Validated Methods). Digital PCR methods performed equally or better than the qPCR methods. Optimized ddPCR methods confirm their suitability for GMO determination in food and feed. 相似文献
9.
Tom Ruttink Rolinde Demeyer Elke Van Gulck Bart Van Droogenbroeck Maddalena Querci Isabel Taverniers Marc De Loose 《Analytical and bioanalytical chemistry》2010,396(6):2073-2089
Competent laboratories monitor genetically modified organisms (GMOs) and products derived thereof in the food and feed chain
in the framework of labeling and traceability legislation. In addition, screening is performed to detect the unauthorized
presence of GMOs including asynchronously authorized GMOs or GMOs that are not officially registered for commercialization
(unknown GMOs). Currently, unauthorized or unknown events are detected by screening blind samples for commonly used transgenic
elements, such as p35S or t-nos. If (1) positive detection of such screening elements shows the presence of transgenic material and (2) all known GMOs are
tested by event-specific methods but are not detected, then the presence of an unknown GMO is inferred. However, such evidence
is indirect because it is based on negative observations and inconclusive because the procedure does not identify the causative
event per se. In addition, detection of unknown events is hampered in products that also contain known authorized events.
Here, we outline alternative approaches for analytical detection and GMO identification and develop new methods to complement
the existing routine screening procedure. We developed a fluorescent anchor-polymerase chain reaction (PCR) method for the
identification of the sequences flanking the p35S and t-nos screening elements. Thus, anchor-PCR fingerprinting allows the detection of unique discriminative signals per event. In addition,
we established a collection of in silico calculated fingerprints of known events to support interpretation of experimentally
generated anchor-PCR GM fingerprints of blind samples. Here, we first describe the molecular characterization of a novel GMO,
which expresses recombinant human intrinsic factor in Arabidopsis thaliana. Next, we purposefully treated the novel GMO as a blind sample to simulate how the new methods lead to the molecular identification
of a novel unknown event without prior knowledge of its transgene sequence. The results demonstrate that the new methods complement
routine screening procedures by providing direct conclusive evidence and may also be useful to resolve masking of unknown
events by known events. 相似文献
10.
S. Del Gaudio A. Cirillo G. Di Bernardo U. Galderisi M. Cipollaro 《Analytical and bioanalytical chemistry》2010,396(6):2135-2142
DNA is widely used as a target for GMO analysis because of its stability and high detectability. Real-time PCR is the method
routinely used in most analytical laboratories due to its quantitative performance and great sensitivity. Accurate DNA detection
and quantification is dependent on the specificity and sensitivity of the amplification protocol as well as on the quality
and quantity of the DNA used in the PCR reaction. In order to enhance the sensitivity of real-time PCR and consequently expand
the number of analyzable target genes, we applied a preamplification technique to processed foods where DNA can be present
in low amounts and/or in degraded forms thereby affecting the reliability of qualitative and quantitative results. The preamplification
procedure utilizes a pool of primers targeting genes of interest and is followed by real-time PCR reactions specific for each
gene. An improvement of Ct values was found comparing preamplified vs. non-preamplified DNA. The strategy reported in the
present study will be also applicable to other fields requiring quantitative DNA testing by real-time PCR. 相似文献
11.
Pengyu Zhu Wei Fu Chenguang Wang Zhixin Du Kunlun Huang Shuifang Zhu Wentao Xu 《Analytica chimica acta》2016
The possibility of the absolute quantitation of GMO events by digital PCR was recently reported. However, most absolute quantitation methods based on the digital PCR required pretreatment steps. Meanwhile, singleplex detection could not meet the demand of the absolute quantitation of GMO events that is based on the ratio of foreign fragments and reference genes. Thus, to promote the absolute quantitative detection of different GMO events by digital PCR, we developed a quantitative detection method based on duplex digital PCR without pretreatment. Moreover, we tested 7 GMO events in our study to evaluate the fitness of our method. The optimized combination of foreign and reference primers, limit of quantitation (LOQ), limit of detection (LOD) and specificity were validated. The results showed that the LOQ of our method for different GMO events was 0.5%, while the LOD is 0.1%. Additionally, we found that duplex digital PCR could achieve the detection results with lower RSD compared with singleplex digital PCR. In summary, the duplex digital PCR detection system is a simple and stable way to achieve the absolute quantitation of different GMO events. Moreover, the LOQ and LOD indicated that this method is suitable for the daily detection and quantitation of GMO events. 相似文献
12.
Quantitative characterization of nucleic acids is becoming a frequently used method in routine analysis of biological samples, one use being the detection of genetically modified organisms (GMOs). Measurement uncertainty is an important factor to be considered in these analyses, especially where precise thresholds are set in regulations. Intermediate precision, defined as a measure between repeatability and reproducibility, is a parameter describing the real situation in laboratories dealing with quantitative aspects of molecular biology methods. In this paper, we describe the top-down approach to calculating measurement uncertainty, using intermediate precision, in routine GMO testing of food and feed samples. We illustrate its practicability in defining compliance of results with regulations. The method described is also applicable to other molecular methods for a variety of laboratory diagnostics where quantitative characterization of nucleic acids is needed. 相似文献
13.
Caprioara-Buda M Meyer W Jeynov B Corbisier P Trapmann S Emons H 《Analytical and bioanalytical chemistry》2012,404(1):29-42
The reliable quantification of genetically modified organisms (GMOs) by real-time PCR requires, besides thoroughly validated quantitative detection methods, sustainable calibration systems. The latter establishes the anchor points for the measured value and the measurement unit, respectively. In this paper, the suitability of two types of DNA calibrants, i.e. plasmid DNA and genomic DNA extracted from plant leaves, for the certification of the GMO content in reference materials as copy number ratio between two targeted DNA sequences was investigated. The PCR efficiencies and coefficients of determination of the calibration curves as well as the measured copy number ratios for three powder certified reference materials (CRMs), namely ERM-BF415e (NK603 maize), ERM-BF425c (356043 soya), and ERM-BF427c (98140 maize), originally certified for their mass fraction of GMO, were compared for both types of calibrants. In all three systems investigated, the PCR efficiencies of plasmid DNA were slightly closer to the PCR efficiencies observed for the genomic DNA extracted from seed powders rather than those of the genomic DNA extracted from leaves. Although the mean DNA copy number ratios for each CRM overlapped within their uncertainties, the DNA copy number ratios were significantly different using the two types of calibrants. Based on these observations, both plasmid and leaf genomic DNA calibrants would be technically suitable as anchor points for the calibration of the real-time PCR methods applied in this study. However, the most suitable approach to establish a sustainable traceability chain is to fix a reference system based on plasmid DNA. 相似文献
14.
PCR technology for screening and quantification of genetically modified organisms (GMOs) 总被引:28,自引:0,他引:28
Holst-Jensen A Rønning SB Løvseth A Berdal KG 《Analytical and bioanalytical chemistry》2003,375(8):985-993
Although PCR technology has obvious limitations, the potentially high degree of sensitivity and specificity explains why it has been the first choice of most analytical laboratories interested in detection of genetically modified (GM) organisms (GMOs) and derived materials. Because the products that laboratories receive for analysis are often processed and refined, the quality and quantity of target analyte (e.g. protein or DNA) frequently challenges the sensitivity of any detection method. Among the currently available methods, PCR methods are generally accepted as the most sensitive and reliable methods for detection of GM-derived material in routine applications.The choice of target sequence motif is the single most important factor controlling the specificity of the PCR method. The target sequence is normally a part of the modified gene construct, for example a promoter, a terminator, a gene, or a junction between two of these elements. However, the elements may originate from wildtype organisms, they may be present in more than one GMO, and their copy number may also vary from one GMO to another. They may even be combined in a similar way in more than one GMO. Thus, the choice of method should fit the purpose. Recent developments include event-specific methods, particularly useful for identification and quantification of GM content. Thresholds for labelling are now in place in many countries including those in the European Union. The success of the labelling schemes is dependent upon the efficiency with which GM-derived material can be detected. We will present an overview of currently available PCR methods for screening and quantification of GM-derived DNA, and discuss their applicability and limitations. In addition, we will discuss some of the major challenges related to determination of the limits of detection (LOD) and quantification (LOQ), and to validation of methods. 相似文献
15.
《Analytical letters》2012,45(15):3093-3125
ABSTRACT There is an increasing need for analytical methods for the detection of genetically modified organisms (GMOs) detection in food due to the growth of use of GMOs, or their derivatives, in the food industry. This paper aims to briefly introduce the reader to GMOs, to describe the state of the art in detection methods for GMOs, and to provide the reader with the associated terminology, mainly borrowed from molecular biology. The main characteristics of GMO are illustrated and future developments using the biosensor approach for detection are presented. 相似文献
16.
Accreditation of GMO detection laboratories: Improving the reliability of GMO detection 总被引:4,自引:0,他引:4
Jana Žel Katarina Cankar Maja Ravnikar Marjana Camloh Kristina Gruden 《Accreditation and quality assurance》2006,10(10):531-536
Reliable and efficient methods for detecting genetically modified organisms (GMOs) are essential for establishing an effective system for traceability all along the supply chain from seed producers to final consumers. The latter is especially meaningful in European Union and other countries where strict legislations on GMOs were set up. Performance of the methods used in laboratories around the world should be uniform, in order to obtain reliable and comparable results. Accreditation is a suitable system for harmonising procedures in each testing laboratory. In this paper, key elements for the accreditation of molecular biology methods for GMO detection according to ISO/IEC 17025 are described. The procedures described are also valuable for the accreditation of molecular methods for all laboratory diagnostics where qualitative and quantitative characterisation of nucleic acids is needed. 相似文献
17.
A review is presented demonstrating that biospecific interaction analysis, using surface plasmon resonance (SPR) and biosensor technologies is a simple, rapid, and automatable approach to detect genetically modified organisms (GMOs). Using SPR, we were able to monitor in real-time the hybridization between oligonucleotide or polymerase chain reaction (PCR)-generated probes and target single-stranded PCR products obtained by using as substrates DNA isolated from normal or transgenic soybean and maize. This procedure allows a one-step, nonradioactive detection of GMOs. PCR-generated probes are far more efficient in detecting GMOs than are oligodeoxyribonucleotide probes. This is expected to be a very important parameter, because information on low percentage of GMOs is of great value. Determination of the ability of SPR-based analysis to quantify GMOs should be considered a major research field for future studies, especially for the analyses of food supplies. 相似文献
18.
Fábio Cristiano Angonesi Brod Jeroen P. van Dijk Marleen M. Voorhuijzen Andréia Zilio Dinon Luis Henrique S. Guimarães Ingrid M. J. Scholtens Ana Carolina Maisonnave Arisi Esther J. Kok 《Analytical and bioanalytical chemistry》2014,406(5):1397-1410
The ever-increasing production of genetically modified crops generates a demand for high-throughput DNA-based methods for the enforcement of genetically modified organisms (GMO) labelling requirements. The application of standard real-time PCR will become increasingly costly with the growth of the number of GMOs that is potentially present in an individual sample. The present work presents the results of an innovative approach in genetically modified crops analysis by DNA based methods, which is the use of a microfluidic dynamic array as a high throughput multi-detection system. In order to evaluate the system, six test samples with an increasing degree of complexity were prepared, preamplified and subsequently analysed in the Fluidigm system. Twenty-eight assays targeting different DNA elements, GM events and species-specific reference genes were used in the experiment. The large majority of the assays tested presented expected results. The power of low level detection was assessed and elements present at concentrations as low as 0.06 % were successfully detected. The approach proposed in this work presents the Fluidigm system as a suitable and promising platform for GMO multi-detection. 相似文献
19.
New approaches in GMO detection 总被引:3,自引:0,他引:3
Maddalena Querci Marc Van den Bulcke Jana Žel Guy Van den Eede Hermann Broll 《Analytical and bioanalytical chemistry》2010,396(6):1991-2002
The steady rate of development and diffusion of genetically modified plants and their increasing diversification of characteristics,
genes and genetic control elements poses a challenge in analysis of genetically modified organisms (GMOs). 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 a few targets to be analysed at once, no longer meet
the testing requirements. Along with new analytical technologies, new approaches for the detection of GMOs authorized for
commercial purposes in various countries 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) algorithms that allow the
conversion of analytical results into an indication of the presence of individual GMOs potentially present in an unknown sample.
This paper reviews the latest progress made in GMO analysis, taking examples from the most recently developed strategies and
tools, and addresses some of the critical aspects related to these approaches. 相似文献
20.
I. M. J. Scholtens E. J. Kok L. Hougs B. Molenaar J. T. N. M. Thissen H. van der Voet 《Analytical and bioanalytical chemistry》2010,396(6):2213-2227
To improve the efficacy of the in-house validation of GMO detection methods (DNA isolation and real-time PCR, polymerase chain
reaction), a study was performed to gain insight in the contribution of the different steps of the GMO detection method to
the repeatability and in-house reproducibility. In the present study, 19 methods for (GM) soy, maize canola and potato were
validated in-house of which 14 on the basis of an 8-day validation scheme using eight different samples and five on the basis
of a more concise validation protocol. In this way, data was obtained with respect to the detection limit, accuracy and precision.
Also, decision limits were calculated for declaring non-conformance (>0.9%) with 95% reliability. In order to estimate the
contribution of the different steps in the GMO analysis to the total variation variance components were estimated using REML
(residual maximum likelihood method). From these components, relative standard deviations for repeatability and reproducibility
(RSDr and RSDR) were calculated. The results showed that not only the PCR reaction but also the factors ‘DNA isolation’ and ‘PCR day’ are
important factors for the total variance and should therefore be included in the in-house validation. It is proposed to use
a statistical model to estimate these factors from a large dataset of initial validations so that for similar GMO methods
in the future, only the PCR step needs to be validated. The resulting data are discussed in the light of agreed European criteria
for qualified GMO detection methods. 相似文献