Detection of CPV-2
A
pair of primers were designed to
detect CPV-2. The forward primer is CTGTGGGTAATGTTGGTTGTT (5’to 3’), and
the reverse primer is TGGTCTTGATGTTGATGGATG (5’ to 3’). The product’s
length is 1163bp. We used DNAiso
Reagent (Takara, China) to extract the genome and ExTaq DNA Polymerase
(Takara, China) to amplify the gene. The following amplification
procedure was applied:
pre-denaturation at 94 °C for 5 min,
then 35 cycles of denaturation at 94 °C for 30 sec, annealing at 48 °C
for 30 sec and extension at 72 °C for 1 min. The final extension at 72
°C for 7 min was performed after the cycles.
Amplification
and sequencing of VP2 gene
Another
pair of primers were designed to amplify the whole VP2 gene. The forward
primer is CACCAGATCATCCATCAACATC (5’ to 3’), and the reverse primer is
AACCACCCACACCATAAC (5’ to 3’).
The
product is 2293bp in length encompassing the entire VP2
gene.
The
super-fidelity
DNA Polymerase used was 2 × Phanta Max Master Mix (Vazyme, China).
Amplification conditions were performed following the manufacturer’s
instructions: pre-denaturation at 95 °C for 30 sec, then 35 cycles of
denaturation at 95 °C for 15 sec, annealing at 56 °C for 15 sec and
extension at 72 °C for 1.5 min, and a final extension at 72 °C for 5
min.
The PCR product was purified by
EasyPure Quick Gel Extraction Kit
(TransGen,
China) according to the manufacturer’s instructions. Then it was cloned
into pEASY-Blunt Cloning Vector (TransGen, China). The recombinant
vector was transformed into Trans1-T1 Phage Resistant Chemically
Competent Cell (TransGen, China). Positive clones were
screened by blue-white selection and further verified by the PCR test,
thus were sent to a third-party company (Huada Gene, Beijing) for
sequencing.
SequenceAnalysis
and Phylogenetic
Construction
Sequences
were assembled using Seqman (DNASTAR, USA), then the nucleotide
sequences and deduced amino acid sequences were aligned with the
ClustalW
method.
To construct a nucleotide
phylogenetic tree based on the full VP2 gene, MEGA-X was used to find
the best DNA models with
maximum
likelihood (ML) method. The bootstrap values were 1000 to analyze the
confidence level. All strains were analyzed with reference strains
obtained from Genbank. Three CPV-2 vaccine strains
(Genbank
accession number: FJ197847, FJ011098, and FJ011097) and two FPLV vaccine
strains (Genbank accession number: EU498681 and EU498680) were included.
Temporal
and Geographical Distribution Analysis
The
reported Chinese CPV-2 strains between 2014 to 2019 with clear
background were collected either from Genbank or related publications,
along with CPV-2 strains in the study (the CPV-2 strains collected in
2020 were excluded since no other CPV-2 strains were isolated in 2020
currently). These data were subjected to temporal distribution analysis
and geographical distribution analysis.