2.3 | Amplification and sequencing of the VP2 gene
The complete VP2 nucleotide sequence was amplified using the following
primers: VP2-F complete: 5’-GGACAAGTAAAAAGAGAC-3’ and VP2-R complete:
5’-TACAAGTACAATATTTCTATGCTG-3’. The length of the amplified fragment was
1925 bp, which covered the entire 1755 bp open reading frame (ORF) of
VP2. A CPV DNA preserved in the laboratory and sterile water were used
as positive and negative controls, respectively.
The amplified products were purified by a Gel Extraction Kit (Cwbio,
China) and cloned into the pEASY blunt vector (TransGen Biotech Co.,
Ltd, Beijing, China). Then, the positive plasmids were sequenced at
least 3 times by Sangon Biotech (Shanghai) Co., Ltd. The 44 sequences of
the VP2 gene were submitted to GenBank with accession numbers
MN810876-MN810919.
2.4. Phylogenetic analysis
The nucleotide sequences of VP2 and the deduced amino acid sequences
were aligned with 18 reference CPV sequences from GenBank using the
MegAlign program of DNASTAR (DNASTAR, USA). The phylogenetic analyses
were conducted by the neighbor-joining method using Mega 6 software. The
reliability of the phylogenetic tree obtained for the VP2 region was
evaluated by running 1,000 replicates in the bootstrap test.