ISO/TC 34/SC 16 - Horizontal methods for molecular biomarker analysis

This document specifies requirements and recommendations to laboratories that perform extraction of polymerase chain reaction (PCR) quality deoxyribonucleic acid (DNA) from cottonseed, cotton leaf and raw material derived therefrom, that is sufficient for the purpose of PCR analysis. This document is applicable to: a) identifying cotton raw material from which PCR quality DNA can be extracted; b) specifying a method for effective DNA extraction from cotton and cotton-derived raw materials; c) specifying the cotton-specific marker(s) to be used as controls for PCR amplification of DNA. A PCR result obtained from analysis of cottonseed, cotton leaf and to some extant raw materials derived therefrom can only indicate that it is not derived from pure genetically modified organism (GMO)-derived cotton. Admixtures of GMO-derived cotton cannot be detected for cotton fibre and cotton fibre-derived materials. This document does not apply to bulk sampling of the seed, bale or processed fabric and yarn. 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.

This document specifies a procedure for the detection of the DNA transition sequence between the 35S promoter region from cauliflower mosaic virus (P35S) and the neomycin-phosphotransferase gene (nptII) from the Tn5 transposon of Escherichia coli. The P35S-nptII segment is part of a construct which confers resistance to neomycin/kanamycin antibiotics frequently found in genetically modified (GM) plants. The detection method is based on real-time PCR and can be used for qualitative screening purposes. For identification and quantification of a specific GM plant (event) a follow-up analysis has to be carried out. This method 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.

This document specifies minimum requirements and minimum performance criteria for conducting a single-laboratory validation study for qualitative (binary) real-time polymerase chain reaction (PCR) methods applied to the detection of specific DNA sequences present in foods. The document is applicable to any single-laboratory validation of a qualitative real-time PCR method used for the detection of specific DNA sequences in food and food products (e.g. for the detection of genetically modified foodstuffs and for species determination, including species known to produce allergenic proteins). The document does not apply to single laboratory validation of qualitative microbiological real-time PCR methods. The document does not apply to the evaluation of applicability and practicability with respect to the specific scope of the PCR method.

This document specifies procedures for DNA extraction from alfalfa (Medicago sativa) seeds and for the specific detection of the herbicide-tolerant alfalfa events J101 and J163 and the lignin-modified alfalfa event KK179 in crop/plant/seed/grain test samples. The detection methods are based on real-time PCR and are targeting the DNA transition sequences between the alfalfa genome and the respective integrated gene construct. The methods can be applied for direct event-specific identification or as a follow-up analysis, if sequences encoding the promoter of the Figwort mosaic virus (P-FMV), the terminator of the nopaline synthase gene from Rhizobium radiobacter (T-nos), or the construct CTP2/CP4-EPSPS (herbicide tolerance) were detected by screening analyses of test samples. In this document, the methods were validated using ground alfalfa seeds and DNA extracted thereof. The PCR methods are also applicable for the analysis of other matrices such as feed and foodstuffs. The application of these PCR methods requires the extraction of an adequate amount of amplifiable DNA from the relevant matrix.

This document specifies requirements for the biobanking of animal germplasm, e.g. semen, embryos, oocytes, gonads and related tissue, including reception, preparation, quality control, storage and distribution. This document is applicable to animal species for food and agriculture. This document is applicable to all organizations performing biobanking of animal biological material and associated data, such as public or private gene banks and germplasm livestock collections centres. NOTE International, national or regional regulations or requirements, or combinations of them, can also apply to specific topics covered in this document.

This document specifies a method for the identification of single fish and fish fillets to the level of genus or species. It allows the identification of a large number of commercially important fish species using DNA barcoding. This method was validated on raw fish. Laboratory experience indicates additional applicability to processed fish products (e.g. cold smoked, hot smoked, salted, frozen, cooked, fried and deep-fried samples). The described method is usually unsuitable for the analysis of highly processed foods (e.g. tins of fish with highly degraded DNA where the fragment lengths are not sufficient for amplification of the targets). Furthermore, it does not apply to complex fish products containing mixtures of two or more fish species. The identification of fish species is carried out by PCR amplification of either a segment of the mitochondrial cytochrome b gene (cytb) or the cytochrome c oxidase I gene (cox1, syn COI), or both, followed by sequencing of the PCR products and subsequent sequence comparison with entries in databases.

This document provides a list of target sequences that can be used to screen for the presence of genetically modified (GM) material in cotton and cotton products. This document is applicable to cottonseed, cotton leaf, cotton fibre and cotton fibre-derived materials from which sufficiently high-quality PCR amplifiable DNA can be extracted. Methods describing the extraction of DNA from different cotton samples can be found in ISO 5354-1[1]. NOTE 1 The list of target sequences provides guidance for the screening of all currently known GM cotton events and GM cotton events that contain the same DNA sequences. Further guidance on screening of foodstuffs is provided in CEN/TS 16707[2]. NOTE 2 Sampling is outside of the scope of this document. Information on sampling cotton products can be found in ISO 1130:1975[3] and in ASTM D1441-12[4].

This document specifies a real-time polymerase chain reaction (real-time PCR) method for the qualitative detection of pigeon-specific DNA derived from food and feed. The method can be applied to distinguish rock pigeon (Columba livia) from other domestic poultry meats (e. g. goose, duck, quail, pheasant). It requires the extraction of an adequate amount of PCR amplifiable DNA from the relevant matrix. The target sequence is a partial fragment of Columba livia breed Danish Tumbler unplaced genomic scaffold, Cliv_1.0 scaffold114 (i.e. GenBank accession number NW_004973337.1),[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 % confidence at this concentration (LOD95 %).

This document specifies a real-time polymerase chain reaction (real-time PCR) method for the qualitative detection of duck-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 duck material derived from mallard duck (Anas platyrhynchos) and spot-billed duck (Anas zonorhyncha). Cross detection of white-winged duck (Asarcornis scutulata), tufted duck (Aythya fuligula), muscovy duck (Cairina moschata) and Mandarin duck (Aix galericulata) is observed. Mallard duck and muscovy duck are domesticated poultry for food, while spot-billed duck, white-winged duck, tufted duck and Mandarin duck are wild avian. The target sequence is a partial fragment of the Anas platyrhynchos breed pekin duck isolate CAU_Pekin_2.0 Chr1, whole genome shotgun sequence (i.e. GenBank accession number JACEUM010000001.1),[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 % confidence at this concentration (LOD95 %).

This document specifies a procedure for the detection of a DNA sequence of the open reading frame five (ORF V) from cauliflower mosaic virus (CaMV) and a procedure for the detection of the DNA sequence of the nopaline synthase (nos) gene from tumour-inducing (Ti) plasmids of phytopathogenic Rhizobium radiobacter (formerly named Agrobacterium tumefaciens). The procedures can be used in the context of screening for genetically modified crop/plants and their derived products to further clarify a positive PCR result for a specific promoter or terminator of CaMV (P-35S, T-35S), or both, and the nos gene (P-nos, T-nos), respectively. The methods specified in this document will detect and identify naturally occurring CaMV or Rhizobium radiobacter (Ti plasmid) DNA, or both, if present in the sample in the absence of a genetically modified plant event containing the specified target sequences. Both methods are based on the real-time polymerase chain reaction (PCR) and are applicable for the analysis of DNA extracted from foodstuffs and other products such as feedstuffs and seeds/grains. The application of the methods requires the extraction of an adequate amount of amplifiable DNA from the relevant matrix. With appropriate calibration material, the CaMV ORF V or nos copy number, or both, can be estimated and compared, respectively, with the estimated copy number for the promoter (P-35S, P-nos) or the terminator (T-35S, T-nos) sequences, or both. Thereby, conclusions are possible about the presence of an unknown genetically modified organism (GMO) in addition to any detected CaMV DNA or Rhizobium radiobacter Ti plasmid DNA, or both, in a test sample.

This document specifies a real-time polymerase chain reaction (real-time PCR) method for the qualitative detection of goose-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 goose material derived from domestic breeds of swan goose (Anser cygnoides domesticus) and domestic goose (Anser anser domesticus). Cross detection of Anser brachyrhynchus, Anser indicus, Branta canadensis, Cygnus atratus, Cygnus buccinator, Cygnus cygnus, Cygnus olor, Nettapus auritus, Oxyura jamaicensis and Stictonetta naevosa of Anseriformes is expected. The method can be applied to distinguish domestic breeds of swan goose (Anser cygnoides domesticus) and domestic goose (Anser anser domesticus) from domestic chicken, duck and turkey which are the most common adulterants of foie gras.[1] It is also able to differentiate the domestic goose from other high-end domestic poultry meats (quail, pigeon, pheasant). The target sequence is a partial fragment of the Anser cygnoides isolate SCWG-2014 breed Sichuan white goose unplaced genomic scaffold, GooseV1.0 scaffold320 (i.e. GenBank accession number NW_025927981.1)[2], 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 % confidence at this concentration (LOD95 %).

This document specifies a real-time polymerase chain reaction (real-time PCR) method for the qualitative detection of turkey-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 turkey material derived from domestic turkey (Meleagris gallopavo) and wild turkey (Meleagris ocellata). The target sequence is a partial fragment of the Meleagris gallopavo chromosome Z DNA sequence (i.e. GenBank accession number NC_015041.2), 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 % confidence at this concentration (LOD95 %).

This document specifies verification and validation parameters and processes for microarray detection and identification of specific nucleic acid sequences. This document provides recommendations and protocols 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; — criteria for assessing technical performance of the microarray platform: This document is applicable to all methods that use microarrays for detection of nucleic acids. It does not apply to the following protocols: — quantitative measurement; — requirements for sample preparation prior to DNA microarray experiments.

This document defines terms for horizontal methods for molecular biomarker analysis in agriculture and food production.

This document specifies general criteria for development, validation and use of nucleic acid analytical methods based on the isothermal polymerase chain reaction (isoPCR). It provides additional information and guidance for specific isoPCR technologies. This document is applicable to food, feed, plant matrices and their propagules, plant pathogens, and animals in which amplification of a specific biomolecular target sequence is required.

This document specifies general requirements for DNA sequencing method performance in the detection and identification of animal species in food and feed products. Performance requirements are limited to Sanger and next generation sequencing (NGS), including second and third generation sequencing, for analysis of single species products and multispecies products. This document is applicable to DNA sequences for mammals, birds, fish, molluscs, crustaceans, amphibians, reptiles and insects, and to the validation of the applicable methods. Methods for DNA species quantification are not considered under the scope of this document.

This document describes general requirements, procedures and performance criteria for evaluating the content of genetically modified (GM) seeds/grains in a lot by a group testing strategy that includes qualitative analysis of sub-sampled groups followed by statistical evaluation of the results. This document is applicable to group testing strategy estimating the GM content on a percentage seed/grain basis for purity estimation, testing towards a given reject/accept criterion and for cases where seed/grain lots are carrying stacked events. This document is not applicable to processed products. NOTE Description of the use of group testing strategy are available in References [1], [7], [8], [18], [19] and [20].

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.

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 %).

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 %).

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 %).

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 %).

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 %).

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 %).

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 %).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

This International Standard describes a procedure for the detection of a DNA sequence of a construct used to (genetically) enhance the growth of fish commonly found in aquaculture. The genetically modified AquAdvantage Atlantic salmon (Salmo salar) carries this construct and can be detected based on a real-time PCR targeting the border between the growth hormone coding sequence (CS-GHc2) of Oncorhynchus tshawytscha (Chinook salmon) and the antifreeze terminator (T-AFP) of (Macro-) Zoarces americanus (ocean pout). This method can be applied to identify the GM fish or for screening purposes. This part of the ISO 21569 series 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. The application of these methods requires the extraction of an adequate amount of amplifiable DNA from the relevant matrix.

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.

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

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.

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.

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.