2.3. PCR

The PCR reaction mix (50 μL) consisted of 100 ng of DNA, 25 μL of HotStar Taq Master mix (Qiagen GmbH, Hiden, Germany), 1 μL of each primer (10 µM), and up to 50 μL of RNAse/DNase-free water. The target gene for the PCR was the D-loop region of mtDNA of both species. The thermal profile consisted of 15 min at 95 °C; then 35 cycles of 30 s at 94 °C, 1 min at 60 °C, and 1 min at 72 °C; then a final elongation step of 5 min at 72 °C. Amplified PCR products were visualized by means of the QIAxcel Advanced System (Qiagen GmbH, Hiden, Germany), which is a fully automated, sensitive, high-resolution system of capillary electrophoresis. The bovine species was confirmed by the presence of a 147 bp fragment obtained by using the following primers: L8249 For, 5′-CAC AAT CCA GAA CTG ACA C-3′, H8357 Rev, and 5′-GTA GGC TTG GGA ATA GTA CGA-3′ [17]. By contrast, the buffalo species was associated with the presence of a 226 bp fragment, obtained with the following primers: Forward-5′- ACT AGATCA CGA GCT TGATCA CCATGC-3′, and Reverse-5′- GTT ATG TGT GAG CAT GGG CTG ATT GGA-3′ [18].