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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 ). Cross detection of plains zebra (Equus quagga) is observed.
The target sequence is a partial fragment of the Equus asinus breed Dezhou chromosome 18, ASM1607732v2 (i.e. GenBank accession number NC_052194.1)[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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