ISO/TC 34/SC 16/WG 8 - Meat speciation

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 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 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 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 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 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 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 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 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.