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).