2.2 DNA extraction and VP2 sequencing

Viral DNA was extracted from faeces using a commercial kit (QIAmp Fast Stool Kit, Qiagen, Germany) according to the manufacturer’s instructions, or homogenisation/boiling as described previously (Decaro et al., 2006). Supernatants obtained after homogenisation/boiling were diluted 1:10 and 1:20 with nuclease-free water to abolish PCR inhibition, and stored at 4°C prior to PCR testing, which was performed within 7 days of extraction. The complete VP2 region was amplified by conventional PCR using three overlapping primer pairs as previously described, with slight modification (Table 1) (Decaro et al., 2008). Each 50 µL PCR reaction contained MyTaq HS Red reaction buffer 1X (5mM dNTPs, 15 mM MgCl2), 10 mM of each forward and reverse primer, 0.5 U of MyTaq Hot Start DNA polymerase (Bioline, Meridian Life Sciences, Australia) and 10 µL of DNA template. Initial denaturation was performed at 94°C for 1 min, followed by 35-39 cycles of denaturation at 94 °C for 30 s, annealing at 50 or 55 °C for 30 s, polymerisation at 72 °C for 30 s and final extension at 72 °C for 1 min. A negative control comprising PBS subjected to the DNA extraction process was included in each run, alongside a positive control sample comprising feline parvovirus (FPV) DNA. Sanger sequencing of PCR products was performed at a commercial laboratory (Macrogen Seoul, South Korea).