Modular real-time PCR screening assay for common European animal families

A screening assay based on real-time PCR and melt curve analysis was developed to detect DNA from nine common European animal families/species and human. The assay consists of a 10-cycle universal pre-amplification followed by specific nested PCR and was designed to exploit the different melting temperatures (T m) of family/species-specific 12S ribosomal ribonucleic acid and cytochrome b fragments, which are amplified in duplex reactions. Case-related modular application is possible. Beyond determination of the animal family and discrimination from human DNA, evaluation of the melt curve in some cases additionally allows for species determination (e.g. cat vs. lynx). The method presents a quick, flexible and sample-saving approach to assess non-human DNA at low expenses, and it is especially useful in resolution of DNA mixtures.