2.2 Polymerase chain reaction (PCR) and sequencing analysis
Extracted viral nucleic acids were subjected for parvovirus PCR
detection using specific primers targeting the VP2 gene (Supplementary
Table S1) as reported (Mochizuki et al., 1996). The PCR reaction
contained 3 µL of DNA template, 1 µL of each primer (10 µM; VPF and
VPR), 12.5 µL of Gotaq Green Master mix (Promega, USA), and
nuclease-free water to 25 µL. The thermal cycling program was comprised
of an initial 94 0C for 10 min, followed by 35 cycles
of 94 0C for 30 s, 55 0C for 2 min,
and 72 0C for 2 min, and then a final 720C for 10 min. The PCR products were resolved on a
1.5% (w/v) agarose gel and then visualized under UV illumination.
Positive PCR products were purified using a NucleoSpin Extract II
(Macherey-Nagel, Germany) and submitted for commercial bi-directional
Sanger’s sequencing (Macrogen, Korea). Subsequently, the selected CPV-2
PCR positive samples were further subjected for whole genome sequencing
using multiple primer sets, as described previously (Perez et al.,
2014), under the same thermal cycling condition as mentioned above
(Supplementary Table S1).
The generated sequences were aligned by the MAFFT (Multiple Alignment
using Fast Fourier Transform program (https://mafft.cbrc.jp) and
compared to those CPV-2 sequences available in GenBank. These alignments
were then subjected for nucleotide and deduced amino acid sequence
analyses as implemented in the BioEdit software package version 7.2
(http://www.mbio.ncsu.edu)