Species Determination Within the Felidae Family Using mtDNA Minibarcoding.

Wildlife forensic analysis frequently deals with highly degraded DNA samples, including those from tanned hides, processed products of traditional Chinese medicine, and specimens stored in preservation fluids. These samples present significant challenges for traditional DNA barcoding due to the difficulty of amplifying sufficiently long DNA fragments. To overcome this limitation, we designed five primer pairs to amplify short mitochondrial DNA (mtDNA) fragments (~100 base pairs). When aligned collectively, these fragments span approximately 500 bp of the cytochrome C oxidase subunit I (COI) gene, which is one of the most common species-barcoding targets. We subjected the primers to a comprehensive validation process, including in silico analysis and experimental verification using various non-degraded and degraded samples of animal tissues. Here, we demonstrate the efficacy and reliability of our DNA minibarcoding method. By addressing the limitations of traditional DNA barcoding, this method improves the accuracy and success of species identification, thereby supporting wildlife conservation, management, and forensic investigations.