CEN/TC 460/WG 2 - Species analyses using DNA-based methods

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 specifies a real-time PCR procedure for the quantification of the amount of roe deer DNA relative to total mammalian and poultry DNA in meat and meat products.
The results of this assay for roe deer are expressed in terms of roe deer (Capreolus capreolus) haploid genome copy numbers relative to mammalian total haploid genome copy numbers. The content of roe deer can also be expressed as a percentage by mass using gravimetrically prepared calibration material from meat mixtures or model samples.
The method has been previously validated in a collaborative trial and applied to DNA extracted from samples that consist of raw roe deer meat in a raw pig meat background as well as raw and boiled sausages.
The limit of detection of the roe deer PCR has been determined experimentally to be at least 5 target gene copies or 0,03 % roe deer.
The compliance assessment process is not part of this document.

This document specifies a real-time PCR procedure for the quantitation of the amount of equine DNA relative to total mammalian DNA in a raw meat sample.
Results of this equine assay are expressed in terms of equine (Equus genus) haploid genome copy numbers relative to total mammalian haploid genome copy numbers. This assay is specific for representatives of the genus Equus and therefore detects horse, mule, donkey and zebra DNA.
The method has been previously validated in a collaborative trial and applied to DNA extracted from samples that consist of raw horse meat in a raw beef (meat) background.
The limit of detection has been determined experimentally to be at least 9 genomic equivalent copies (~ 17 haploid target gene copies) for both the horse genome (E. caballus) and mammalian genome (raw meat samples) based on the lowest dilution on the respective calibration curves through single laboratory validation. The lowest relative horse content of the target sequence included in the collaborative trial was a mass fraction of 0,1 % based on gravimetrically prepared raw horse muscle tissue in a raw beef muscle tissue background.
The compliance assessment process is not part of this document.

This document describes a procedure for the identification of single fish and fish fillets to the
level of genus or species.
The identification of fish species is carried out by PCR amplification of either a segment of the
mitochondrial cytochrome b gene (cytb) [1] or the cytochrome c oxidase I gene (cox1, syn COI)
[2], [3] or both, followed by sequencing of the PCR products and subsequent sequence
comparison with entries in databases [4], [5]. The methodology allows the identification of a large
number of commercially important fish species.
The decision whether the cytb or cox1 gene segment or both are used for fish identification
depends on the declared fish species, the applicability of the PCR method for the fish species
and the availability of comparative sequences in the public databases.
This method has been successfully validated on raw fish fillets, however, laboratory experience
is available that it can also be applied to processed, e.g. cold smoked, hot smoked, salted,
frozen, cooked, fried, deep fried
samples.
This document 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 is not applicable for complex fish products containing mixtures of two or
more fish species.

This document specifies a method for the identification of meat derived from mammals and birds to the level of genus or species and allows the identification of a large number of commercially important as well as exotic meat species using DNA barcoding.
This method was validated on DNA isolated from single pieces of raw meat. This method can also be used for the identification of single meat animal species in some processed products.
The described method is unsuitable for the analysis of highly processed foods with highly degraded DNA where the fragment lengths are not sufficient for amplification of the targets. Furthermore, it is not applicable for complex meat products containing mixtures of two or more meat species.
The identification of meat 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 specifies a method for the taxonomic identification of a single bivalve or piece of bivalve to the genus or species level using DNA barcoding. It allows the identification of a large number of commercially important bivalve species.
This method was validated on raw mussels. Laboratory experience indicates additional applicability to processed bivalve 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 mussels, with highly degraded DNA where the fragment lengths are not sufficient for amplification of the target. Furthermore, it is not applicable for complex seafood products containing mixtures of two or more bivalve species.
The identification of bivalve species is carried out by PCR amplification of a segment of the mitochondrial 16S rRNA gene, followed by sequencing of the PCR productsand subsequent sequence comparison with entries in databases.