Factors influencing the accuracy of measurements with real-time PCR: The example of the determination of processed animal proteins

Quantification of traces of processed animal proteins (PAPs) in feedingstuffs is an important requirement to enforce upcoming European legislation on the use of these materials in animal nutrition. One of the techniques proposed in this context is real-time polymerase chain reaction (real-time PCR) allowing for the determination of PAPs at various taxonomic levels. A challenge in this field is the fact that real-time PCR measures DNA copy numbers, whereas legislation expresses tolerance limits in terms of mass fractions of PAPs in feedingstuffs. Here, we evaluated the impact of different sterilisation temperatures performed under real world rendering conditions showing that at high temperatures the DNA copy number was significantly reduced. This effect complicates the determination of the PAPs mass fraction in feedingstuffs by means of real-time PCR. In addition we showed that measuring at trace levels could lead to a high variation of the measured DNA copy number, characterised by a positively skewed distribution. Various implications of these findings are discussed.