Carnivore Protoparvovirus 1 VP2 Gene Sequencing

DNA was extracted for sequencing of the Carnivore protoparvovirus 1 VP2 gene from tissue using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) or from faecal samples using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Germany) or by homogenisation, boiling and centrifugation, as previously described [11 (link)]. The extracted DNA was amplified by conventional PCR using 5 u/µL of My Taq HS Red DNA polymerase (Bioline, USA), 1–90 ng of DNA, 1 x MyTaq Red reaction buffer and a final primer concentration of 0.2 µM in a final reaction volume of 25 µL. Three sets of overlapping primers were used to amplify the entire VP2 region (1931 bp) as described previously, with minor modifications (Table 1) [12 (link)]. An initial denaturation step at 94 °C for 1 min, followed by 35–40 cycles of denaturation at 94 °C for 30 s, annealing at 50 or 55 °C for 30 s and extension at 72 °C for 30 s, ending with a final extension at 72 °C for 1 min, was used for DNA amplification. The PCR products were separated by electrophoresis on a 1% agarose gel (Bio-Rad Laboratories, Hercules, CA, USA) in 1× tris-acetate EDTA and visualized using SYBR Safe DNA (Invitrogen, Carlsbad, CA, USA). Sanger sequencing of positive PCR products was performed commercially (Macrogen, Seoul, Korea).