Development of a real-time PCR approach for the relative quantitation of horse DNA

Abstract

The 2013 European-wide issue involving the undeclared presence of horse meat in beef products emphasised the need for the development of accurate analytical approaches for the quantitative detection of meat adulteration. As part of a UK Government, Department for Environment, Food & Rural Affairs (Defra) project, a real-time PCR method was developed for the quantitation of horse DNA relative to the total amount of mammalian DNA present in raw meat samples. Single copy nuclear DNA targets were chosen and assays selected that targeted an equine growth hormone receptor and a mammalian/poultry myostatin gene. The method was challenged against a range of gravimetrically prepared raw horse meat in raw beef ad-mixtures, and demonstrated good performance characteristics, including high mean r-squared values (>0.995) and PCR efficiencies (>90%). The limit of detection was estimated at less than five horse genome equivalents, and the limit of quantitation to be ≤0.1% w/w gravimetric preparation of raw horse meat in a raw beef meat background. Assessment of multiple 1% w/w raw gravimetric samples estimated the mean and analytical measurement uncertainty (based on a 95% confidence interval) to be 1.58 ± 0.54% w/w, thereby demonstrating good trueness and precision. The method was validated for DNA extracted from samples that consisted of raw horse meat in a raw beef meat background. The development and publication of this non-proprietary novel real-time PCR method for the quantitation of horse DNA will inform and strengthen the decision making processes of food companies and regulators.