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.