This document specifies a procedure for the detection of a DNA sequence present in a genetically modified linseed (Linum usitatissimum) line (event FP967, also named as “CDC Triffid”). For this purpose, extracted DNA is used in a real-time PCR and the genetic modification (GM) is specifically detected by amplification of a 105 bp DNA sequence representing the transition between the nopaline synthase gene terminator (Tnos) from Agrobacterium tumefaciens and the dihydrofolate reductase gene (dfrA1) from a Class 1 integron of Escherichia coli. The method described is applicable for the analysis of DNA extracted from foodstuffs. It can also be suitable for the analysis of DNA extracted from other products such as feedstuffs and seeds. The application of this method requires the extraction of an adequate amount of amplifiable DNA from the relevant matrix for the purpose of analysis.

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This document specifies a real-time polymerase chain reaction (real-time PCR) method for the qualitative detection of donkey-specific DNA derived from food and feed. It requires the extraction of an adequate amount of PCR amplifiable DNA from the relevant matrix and can be applied to the detection of donkey material derived from donkey (Equus asinus), mule (Equus caballus ♀ × Equus asinus ♂) and hinny (Equus caballus ♂ × Equus asinus ♀). The assay also detects the species zebra (Equus burchellii). The target sequence is a partial fragment of the Equus asinus isolate Maral har breed Guanzhong donkey unplaced genomic scaffold, ASM130575v1 scaffold786, whole genome shotgun sequence (i.e. GenBank accession number NW_014638576.1)[1], which is present as a single copy per haploid genome. The provided PCR assay for this target has an absolute limit of detection of five copies per reaction, with ≥ 95 % replicability at this concentration (LOD95 %).

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This document specifies a real-time polymerase chain reaction (real-time PCR) method for the qualitative detection of goat-specific DNA derived from food and feed. It requires the extraction of an adequate amount of PCR amplifiable DNA from the relevant matrix and can be applied to the detection of goat material derived from goat (Capra hircus). The target sequence is a partial fragment of the goat chromosome 9 DNA sequence (i.e. GenBank accession number NC_030816.1)[1], which is present as a single copy per haploid genome. The provided PCR assay for this target has an absolute limit of detection of five copies per reaction, with ≥ 95 % replicability at this concentration (LOD95 %).

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This document specifies a real-time polymerase chain reaction (real-time PCR) method for the qualitative detection of bovine-specific DNA derived from food and feed. It requires the extraction of an adequate amount of PCR amplifiable DNA from the relevant matrix and can be applied to the detection of bovine material derived from cattle including taurine (Bos taurus) and zebu (Bos indicus). The assay also detects the species bison (Bison bison) and yak (Bos mutus). The target sequence is a partial fragment of the bovine nuclear beta actin gene in chromosome 25 (i.e. GenBank accession number NC_037352.1)[1][2][3], which is present as a single copy per haploid genome. The provided PCR assay for this target has an absolute limit of detection of five copies per reaction, with ≥ 95 % replicability at this concentration (LOD95 %).

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This document specifies a real-time polymerase chain reaction (real-time PCR) method for the qualitative detection of chicken-specific DNA derived from food and feed. It requires the extraction of an adequate amount of PCR amplifiable DNA from the relevant matrix and can be applied to the detection of chicken material derived from chicken (Gallus gallus domesticus) and jungle fowl (Gallus gallus). The target sequence is a partial fragment of the Gallus gallus transforming growth factor beta 3, intron 4 (TGF-β3) gene (i.e. GenBank accession number AY685072.1)[1], which is present as a single copy per haploid genome. The provided PCR assay for this target has an absolute limit of detection of five copies per reaction, with ≥ 95 % replicability at this concentration (LOD95 %).

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This document specifies a real-time polymerase chain reaction (real-time PCR) method for the qualitative detection of porcine-specific DNA derived from food and feed. It requires the extraction of an adequate amount of PCR amplifiable DNA from the relevant matrix and can be applied to the detection of porcine material derived from pig (Sus scrofa domesticus) and wild boar (Sus scrofa). The target sequence is a partial fragment of the Sus scrofa beta actin (ACTB) gene, partial cds. (i.e. GenBank accession number DQ452569.1)[1], which is present as a single copy per haploid genome. The provided PCR assay for this target has an absolute limit of detection of five copies per reaction, with ≥ 95 % replicability at this concentration (LOD95 %).

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This document specifies a real-time polymerase chain reaction (real-time PCR) method for the qualitative detection of ovine-specific DNA derived from food and feed. It requires the extraction of an adequate amount of PCR amplifiable DNA from the relevant matrix and can be applied to the detection of ovine material derived from sheep (Ovis aries). The target sequence is a partial fragment of the ovine nuclear prolactin receptor short form mRNA gene (PRLR) (i.e. GenBank accession number AF041979.1)[1], which is present as a single copy per haploid genome. The provided PCR assay for this target has an absolute limit of detection of five copies per reaction, with ≥ 95 % replicability at this concentration (LOD95 %).

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This document specifies a real-time polymerase chain reaction (real-time PCR) method for the qualitative detection of horse-specific DNA derived from food and feed. It requires the extraction of an adequate amount of PCR amplifiable DNA from the relevant matrix and can be applied to the detection of horse material derived from domestic horse (Equus caballus), mule (Equus caballus ♀ × Equus asinus ♂), hinny (Equus caballus ♂ × Equus asinus ♀) and zebroid (Equus caballus × Equus simplicidens). The assay also detects the species Przewalski's horse (Equus przewalskii) and zebra (Equus burchellii). The target sequence is an Equus caballus isolate Twilight breed thoroughbred chromosome 28, EquCab3.0, whole genome shotgun sequence (i.e. GenBank accession number NC_009171.3)[1], which is present as a single copy per haploid genome. The provided PCR assay for this target has an absolute limit of detection of five copies per reaction, with ≥ 95 % replicability at this concentration (LOD95 %).

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This document describes a procedure for the detection of the DNA transition sequence between the 35S promotor (P35S) from Cauliflower mosaic virus and a modified phoshinothricin-acetyltransferase gene (pat) from Streptomyces viridochromogenes. The P35S-pat construct is frequently found in genetically modified plants with tolerance for phosphinothricin-containing herbicides. The P35S-pat construct specific method is based on a real-time PCR and can be used for qualitative and quantitative screening purposes. For identification and quantification of a specific event, a follow-up analysis can be carried out. This document is applicable to the analysis of DNA extracted from foodstuffs. It can also be suitable for the analysis of DNA extracted from other products such as feedstuffs and seeds. The application of this method requires the extraction of an adequate quantity and quality of amplifiable DNA from the relevant matrix.

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This document specifies performance criteria for immunochemical methods for the detection and/or quantification of a specific protein or protein(s) of interest [POI(s)] in a specified matrix. The methods discussed are applicable to the analysis of proteins from a variety of sample types. Some uses for these methods include, but are not limited to, analysing proteins involved in crop and food production, food processing, food marketing, food safety, biotechnology or disease indexing.

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This document specifies minimum requirements of performance characteristics for the detection of nucleic acid sequences (DNA) by molecular methods, such as the polymerase chain reaction (PCR), including different post-PCR detection methods, real-time PCR, single and/or multiple probe-based detection techniques as well as the combination of such methods. The document is applicable to the detection, identification and quantification of DNA from animal species of higher and lower taxonomic groups in foodstuffs, and the validation of applicable methods. It is applicable to mammals, birds, reptiles, amphibians, fishes, molluscs, crustaceans and insects. Typical examples for each are listed in Annex A.

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This document provides requirements and recommendations to laboratories that perform genetically modified organism (GMO) analyses in cottonseed, leaf, cotton fibre and cotton fibre-derived materials. The following are within the scope of this document: a) identifying the materials to be assessed, based on the probability of obtaining good quality, fit for purpose DNA from the materials in subsequent steps in the cotton cloth production process; b) specifying a method for efficient DNA isolation from cotton and cotton-derived materials described under point a); c) specifying the cotton-specific method(s) to be used as control for amplifiable DNA; d) specifying the screening procedure that provides optimal chances to detect GMOs as a result of the performance of the lowest number of genetically modified (GM) element screening assays. NOTE 1 The protocol allows for the screening of all currently known GM cotton events and is set up in a way that optimizes the probability of also detecting unknown GM cotton events that possibly contain similar DNA sequences. Further information is given in CEN/TS 16707. Sampling is outside of the scope of this document. NOTE 2 A recommended sampling method is given in ISO 6497. General guidance for the sampling of bulk materials or for cotton-based products is available in standards such as ASTM D1441‑12 and CEN/TS 15568.

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This document specifies methods that yield a binary result and are used for the determination in food or food products (including seeds of food crops) of the presence of molecular biomarkers. These methods are typically used where the measurand is expected to be present in very small amounts and concentrations at the limit of detection (LOD). Methods are validated in terms of the probability of detection (POD) and of the precision of the POD. They do not rely on the concept of false positive/false-negative results, or the concept of LOD. However, inferences about the precision of the classical LOD can be made. This document describes the extent of method validation. The annexes provide different statistical models that can be considered depending on the analytical method, structure of data and statistical experience. This document does not apply to quantitative methods that are used to make a detection decision by comparing the value of a response to a cut-off value using a quantitative method, where the methods are validated by using quantitative statistics on the responses. This document also does not apply to microbiological test methods, starch, essential oils or quantitative methods.

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ISO/TS 21569-5:2016 specifies a procedure for the detection of a DNA sequence used in genetically modified (GM) plants by means of a real-time PCR (polymerase chain reaction). The method detects a 78 base pairs long segment of the Figwort mosaic virus 34S promoter DNA sequence. This segment in some GM plants is indicated as FMV promoter (P-FMV) and in other GM plants as FMV enhancer (E-FMV). The method was developed and validated for the analysis of DNA extracted from foodstuffs. It may be suitable also for analysis of other products such as feedstuffs and seeds. The procedure requires the extraction of an adequate quantity and quality of amplifiable DNA from the test sample. The DNA sequence amplified by the P-FMV element-specific method can be detected in samples which contain DNA of the naturally occurring Figwort mosaic virus. For this reason, it is necessary to confirm a positive screening result. Further analyses are required using construct-specific or event specific methods.

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ISO/TS 21569-4:2016 specifies a procedure for the detection of a DNA sequence of the promoter region of the nopaline synthase gene (P-nos) from Agrobacterium tumefaciens and a procedure for the detection of the DNA transition sequence between P-nos and the neomycin-phosphotransferase gene (nptII) from the Tn5 transposon of Escherichia coli K12. The nos-promoter and the P-nos-nptII-construct are frequently found in genetically modified plants. The P-nos and P-nos-nptII specific methods are based on real-time PCR and can be used for qualitative screening purposes. For identification and quantification of a specific genetically modified plant (event) a follow-up analysis has to be carried out. The methods described are applicable for the analysis of DNA extracted from foodstuffs. They may also be suitable for the analysis of DNA extracted from other products such as feedstuffs and seeds. The application of these methods requires the extraction of an adequate amount of amplifiable DNA from the relevant matrix. The DNA sequence amplified by the P-nos element-specific method can be detected in samples which contain DNA of the naturally occurring Ti-plasmid of A. tumefaciens. For this reason, it is necessary to confirm a positive screening result. Further analyses are required using construct-specific or event specific methods.

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ISO/TS 21569-6:2016 specifies a procedure for the detection of a DNA sequence of the modified cry1Ab/Ac gene and a procedure for the detection of the DNA transition sequence between the maize ubiquitin promoter (Pubi) and the cry1Ab/Ac gene. The modified cry1Ab/Ac gene and the Pubi-cry construct are frequently found in genetically modified Bt plants. Both detection methods are based on real-time PCR and can be used for qualitative screening purposes. For identification and quantification of a specific genetically modified plant (event) a follow-up analysis has to be carried out. ISO/TS 21569-6:2016 is applicable for the analysis of DNA extracted from foodstuffs. It may also be suitable for the analysis of DNA extracted from other products such as feedstuffs and seeds. The application of these methods requires the extraction of an adequate amount of amplifiable DNA from the relevant matrix.

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ISO 16577:2016 gives the definition of terms used in the International Standards published in the frame of ISO/TC 34/SC 16.

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The methods and SSR markers included in ISO/TR 17622:2015 can be used for testing hybrid conformity and other applications such as molecular fingerprinting of varieties and checking variety identity.

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The methods and SSR markers included in ISO/TR 17623:2015 are suitable for applications such as testing hybrid conformity, molecular fingerprinting of varieties, and checking variety identity.

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ISO 16578:2013 defines terms for the detection of nucleic acid sequence of interest using DNA microarrays for detection of nucleic acid. It specifies the verification processes and parameters for molecular biology analysis, including the detection and identification of specific nucleic acid sequences. ISO 16578:2013 provides recommendations and protocol for microarray design and manufacture, validation of hybridization specificity, interlaboratory validation of qualitative methods, determination of limits of detection for a microarray, determination of range of reliable signals, and criteria to assessing technical performance of the microarray platform.

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ISO 13495:2013 specifies molecular tools for generating molecular profiles of varieties of plant species, enabling varietal identification, i.e. confirmation of identity in relation to one or more references. ISO 13495:2013 is applicable to various matrices, seeds, leaves, roots, industrial products composed of only one variety. Matrices presented in the form of mixtures of varieties (such as purees, compotes, flours) are excluded from the scope of ISO 13495:2013. It does not deal with genetic purity.

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ISO 24276:2006 specifies how to use the standards for sampling strategies (EN/TS 21568), nucleic acid extraction (ISO 21571), qualitative nucleic acid analysis (ISO 21569) and quantitative nucleic acid analysis (ISO 21570), and their relationship in the analysis of genetically modified organisms in foodstuffs, and contains general definitions, requirements and guidelines for laboratory set-up, method validation requirements, description of methods and test reports. It has been established for food matrices, but could also be applied to other matrices (e.g. seeds, feed and plant samples from the environment).

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ISO 21570:2005 provides the overall framework of quantitative methods for the detection of genetically modified organisms (GMO) in foodstuffs, using the polymerase chain reaction (PCR). It defines general requirements for the specific amplification of DNA target sequences in order to quantify the relative GMO-derived DNA content and to confirm the identity of the amplified DNA sequence. Guidelines, minimum requirements and performance criteria laid down in ISO 21570:2005 are intended to ensure that comparable, accurate and reproducible results are obtained in different laboratories. ISO 21570:2005 has been established for food matrices, but is also applicable to other matrices, e.g. feed and plant samples from the environment.

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ISO 21569:2005 describes the procedure to qualitatively detect genetically modified organisms (GMOs) and derived products by analysing the nucleic acids extracted from the sample under study. The main focus is on polymerase chain reaction (PCR) based amplification methods. It gives general requirements for the specific detection and identification of target nucleic acid sequences (DNA) and for the confirmation of the identity of the amplified DNA sequence. Guidelines, minimum requirements and performance criteria laid down in ISO 21569:2005 are intended to ensure that comparable, accurate and reproducible results are obtained in different laboratories. ISO 21569:2005 has been established for food matrices, but could also be applied to other matrices (e.g. feed and plant samples from the environment). Specific examples of methods are provided in Annexes A to D.

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ISO 21571:2005 provides general requirements and specific methods for DNA extraction/purification and quantification. These methods are described in Annexes A and B. ISO 21571:2005 has been established for food matrices, but could also be applicable to other matrices, such as grains and feed. It has been designed as an integral part of nucleic-acid-based analytical methods, in particular ISO 21569 on qualitative analytical methods, and ISO 21570 on quantitative analytical methods.

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ISO/TS 21569-3:2015 describes a procedure for the detection of the DNA transition sequence between the 35S promotor (P35S) from Cauliflower mosaic virus and a modified phoshinothricin-acetyltransferase gene (pat) from Streptomyces viridochromogenes. ISO/TS 21569-3:2015 is applicable for the analysis of DNA extracted from foodstuffs. It may also be suitable for the analysis of DNA extracted from other products such as feedstuffs and seeds.

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ISO 21572:2013 provides general guidelines and performance criteria for methods for the detection and/or quantification of specific proteins or protein(s) of interest [POI(s)] in a specified matrix. These general guidelines address existing antibody based methods. Methods other than those described in Annex A or Annex B can also detect the POI. The same criteria as outlined in ISO 21572:2013 apply generally.

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This method describes a procedure for the detection of a DNA sequence present in a genetically modified linseed (Linum usitatissimum) line (event FP967, also named as "CDC Triffid"). For this purpose, extracted DNA is used in a real-time PCR and the genetic modification (GM) is specifically detected by amplification of a 105 bp DNA sequence representing the transition between the nopalin synthase gene terminator (Tnos) from Agrobacterium tumefaciens and the dihydrofolate reductase gene (dfrA1) from a Class 1 integron of Escherichia coli. The method described is applicable for the analysis of DNA extracted from foodstuffs. It may also be suitable for the analysis of DNA extracted from other products such as feedstuffs and seeds. The application of this method requires the extraction of an adequate amount of amplifiable DNA from the relevant matrix for the purpose of analysis.

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ISO/TS 21098:2005 defines the principles and specifies the nature of the information to be supplied for acceptance of a method as an annex to ISO 21569, ISO 21570 or ISO 21571. It also specifies the process for adding, amending and retaining methods annexed to these standards. ISO/TS 21098:2005 is necessary in order to attain consistency in methods that are to be employed as part of the standards. It does not cover the specifics of the development of a method or laboratory set-up.

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ISO 21572:2004 provides general guidelines and performance criteria for methods for the detection and/or quantitation of specific proteins derived from genetically modified (GM) plant material in a specified matrix. These general guidelines address existing antibody based methods. Methods other than those described in annex A may also detect the protein. The same criteria as outlined in this standard generally apply.

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