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Standards Development
The method described uses real-time PCR to quantitate the amount of equine DNA relative to the amount of total mammalian DNA extracted from a raw beef (meat) sample. The current method has been applied to and validated for DNA extracted from samples that consist of raw horse meat in a raw beef (meat) background on a gravimetric (w/w) basis. Test samples containing equine DNA in a background of beef DNA are analysed by a relative quantitation approach utilising singleplex real-time PCR assays targeting the equine growth hormone receptor gene and the myostatin gene which is present in mammals and poultry. DNA template concentration is quantified prior to undertaking the real-time PCR in order to enable test input levels to be normalised appropriately. 100% horse DNA derived from authenticated raw horse meat materials that have been extracted and treated in the same manner to the test samples should be used to generate separate calibration curves for both the horse specific target and the mammalian target based on estimated genome equivalents. Test samples are evaluated using the same equine specific and universal mammalian real-time PCR assays. Estimated genome equivalent copy numbers are determined for the test samples using the equine and mammalian calibration curves. The percentage equine DNA content of the test sample is expressed as a ratio of the number of horse genome equivalents relative to the total mammalian genome equivalents present in the sample.
During the UK/EU Horse-meat incident of 2013 beef products were found to be adulterated with horse meat. In order to support current food labelling regulations, it is necessary to be able to identify and quantify levels of meat species food components in relation to labelling thresholds. A reliable molecular method for both species identification and quantification is therefore required in order to avoid similar fraudulent activities. A real-time PCR approach for the relative quantitation of equine DNA, with a limit of detection of 5 genome equivalents per test and a demonstrable acceptable level of precision for enforcement action provides a robust and repeatable solution for this issue.
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