Mytaq hs polymerase mix

PCR reactions using MyTaq HS polymerase mix (Bioline, Luckenwalde, Germany), 200 nM primers, and approximately 200 ng of the extracted template DNA were performed [24 (link)]. Screening of herpesviruses using nested PCR targeting a partial sequence of the DNA-dependent DNA polymerase gene (Pol, UL30) was performed as described previously [28 (link)]. Amplification and sequencing of the Pol, gB, UL49.5, ORF15 and genes encoded in the unique short segment (ORF69–ORF74; protein kinase, gG, gp2, gD, gI, and gE) was performed using primers specific to EHV-1 and EHV-9. The amplified products were purified and directly sequenced. The primers for nested PCR amplification, ORF15 and UL49.5 have been described previously [24 (link)]. New primers used in this study are listed in Table S2. The nucleotide sequences obtained in this study have been deposited in GenBank (accession numbers: KX101085–KX101111).

PCR products were amplified from genomic DNA from multiple bear tissues using the following primer pairs UrsusERV-F1 (5′-AGCCTACTTGGGATGACTGC-3′), UrsusERV-R1 (5′-GAGACCAGCCACTAGAGCCT-3′) and UrsusERV-F2 (5′-TAGAAGGGAGACAGATGAGGA-3′), UrsusERV-R2 (5′-TGAGAGTTATCCTGGGCTCA-3′). Polymerase chain reactions were performed in 50 μL reaction volumes containing 0.5 U of MyTaq HS polymerase mix (Bioline, Taunton, MA, USA), 400 nM of either paired primers, and 1 µL of DNA template. Thermocycling conditions were 95 °C denaturing for 3 min followed by 35 cycles of 95 °C for 15 s, 62 °C for 20 s, 72 °C for 15 s. Amplified products were visualized on 1.5% (w/v) agarose gels using Midori Green gel stain (Nippon Genetics, Dueren, Germany). Amplification products were sequenced using the forward and reverse primer used in the amplification (LGC Genomics, Berlin, Germany).