Detection and quantification of meat species by qPCR in heat-processed food containing highly fragmented DNA

Analytical methods for the detection and quantification of species in processed food are not yet fully established due to the negative effects of heat treatments on the targeted proteins and DNA, which compromises immuno- and PCR-based detection methods. Here, real-time PCR (qPCR) detectors based on Minor Groove Binding (MGB) probes were used to test their ability to detect and quantify pork DNA in beef/pork meat binary mixtures subjected to cooking and sterilization. A single-point matrix standard strategy allowed calibration of qPCR results for direct quantification of meat proportions in binary mixture. The correlation between heat treatment and the extent of DNA degradation was also experimentally tested. Cooking at 65 °C followed by sterilization at 126 °C from 10 to 30 min led to DNA rupture to approximately 100 bp-long fragments, which still allowed detection of 5% pork and its accurate quantification in binary mixtures. The results demonstrate the capability of short qPCR detectors for quantification of meat products in processed food, and illustrate the necessity to include matrix-adapted standards in the assay.