Influences on the sensitivity of real-time PCR for the detection of bovine DNA in heat-sterilised feedstuffs

The present study evaluated two previously developed methods for amplification of bovine mtDNA segments of 109 and 271 base pairs (bp) by real-time polymerase chain reaction (PCR). Beef samples were sterilised experimentally at different temperatures (126 degrees C, 129 degrees C, 132 degrees C and 135 degrees C). These experimentally sterilised beef samples and nine commercial meat and bone meals (MBM) were mixed to a reference plant concentrate in strengths of 50%, 10%, 5%, and 1%. The results of the following PCR showed that the Bos-109 real-time PCR assay was able to detect all the experimental beef samples with exception of the mixtures of beef heated experimentally to 135 degrees C. In mixtures of industrial MBM bovine DNA were always found. Comparatively, the beef sterilised at 135 degrees C and 132 degrees C (and their respective mixtures) and the mixture containing 1% of beef sterilised at 129 degrees C were not detectable with the PCR assay amplifying a target of 271 bp. Using this PCR mixtures of industrial MBM were only weakly detected. The low concentrated mixtures of the extremely processed MBM-1 and MBM-2 even reported negative. These results indicate that the detectability of bovine DNA is strongly influenced by the degree of the thermal treatment. Only the PCR assay amplifying relatively short fragments of a multi-copy mitochondrial target was reliable for the detection of correctly heated MBM in mixed feed.