Multilevel D-loop PCR identification of hunting game

The control region of mtDNA (D-loop) was used for hair samples of the five hunting game species identification: red deer (Cervus elaphus), roe deer (Capreolus capreolus), fallow deer (Dama dama), mouflon (Ovis aries musimon), and wild boar (Sus scrofa). For D-loop multilevel PCR detection scheme was applied in six primers (CE CVZV 1=5′-GATCACGAGCTTGATCACCA-3′; CE CVZV 2=5′-AGGAGTGGGCGATTTTAGGT-3′; DD CVZV 3=5′-CGCGTGAAACCAACAACCCGC-3′; DD CVZV 4=5′-CCGGGTCGGGGCCTTAGACG-3′; SSW CVZV 5=5′-ACACGTGCGTACACGCGCATA-3′; SSW CVZV 6=5′-GGTGCCTGCT T TCGTAGCACG-3′) designed to identify unknown biological samples of the hunting game animals. The PCR reaction volume was 25μl at conditions 95°C for 2min, 94°C for 30s, 60°C for 30s, 72°C for 30s, 35cycles, with last extension at 72°C for 10min. D-loop mtDNA amplicons of the game animals are characterized with specific PCR product sizes depending on species: red deer=163bp and 140bp, fallow deer=280bp and 138bp, roe deer=303bp, 280bp, 160bp and 138bp, mouflon=299bp and 178bp, wild boar=137bp and 229bp. •Multilevel PCR was used for the identification of five hunting game species.•Six specific primers were designed to analyse D-loop of the five game species.•Technology is suitable for detection of unknown biological samples from wildlife.•The oligonucleotides sequences were registered in www.boldsystems.org.