The risk of
bovine spongiform encephalopathy propagation was drastically reduced after the European Union (EU) Health Authorities adopted restrictions involving a ban on animal-derived
proteins in the diet of farm animals. Currently, the EU's officially recommended method for controlling meat and
bone meal (MBM) in animal feed is the microscopic method, which involves the identification of bone fragments on the basis of their morphological characteristics. Recently, we demonstrated that a polymerase chain reaction (PCR)-based assay can be used for the detection of taxon-specific
DNA in MBM and animal feeds. To ensure the safe rendering of animal by-products, the EU Council requires that this material be treated at 133 degrees C at 300 kPa for 20 min. Here we investigate the relationship between
DNA degradation, PCR amplification, and MBM heat treatment. With a competitive PCR-based approach, we compare the amplification efficiency of bovine
mitochondrial DNA target sequences of different lengths in several heat-treated MBM samples. For our method, a synthetic competitive
DNA is used as an internal control for both
DNA extraction and PCR reaction. A correlation between an increase in treatment temperature and a reduction in the size of the target sequences suitable for amplification was observed, suggesting progressive DNA fragmentation due to the temperature. We show that short amplicons (147 bp) can be used to detect the presence of bovine
mtDNA in MBM samples treated according to the current European regulations. The use of such a competitive approach to compare amplification efficiency levels of targets of different lengths might represent a useful tool for the determination of both the amount of MBM in animal feeds and its proper heat treatment.