Double specific nested PCR and diagnostic SNP assay for species identification in lynx fecal critical samples

Abstract

The single lynx species present in the Iberian Peninsula—Lynx pardinus (Temmink, 1827)—is one of the most threatened felines in the world so, any biological indication of its presence is significant. However, it is a very elusive species and physical evidence through direct visualization or camera trapping is difficult to obtain. Genetic noninvasive methods, in particular, molecular scatology, are a reliable alternative to unequivocally identify the presence of a species in a given geographical area. We have developed a highly specific and sensitive molecular method to identify the presence of this critically endangered lynx species from severely deteriorated fecal samples. The method consists in a double specific nested PCR of mitochondrial D-loop sequences and two diagnostic SNPs detected by a multiplex primer extension reaction.