Sequencing and phylogenetic analysis of the complete genome of
the Bo/CH/HB/BD/2019 strain
Based on the GenBank sequences, we designed six pairs of
specific primers using Primer Premier 5 software. The reference
sequences were AY126472.2, JX145650.1, EU794907, AF097917.5, MK159169.1
and NC_029645.1. The complete BNoV genome of the strain
Bo/CH/HB/BD/2019 was amplified using six sequence-specific primer pairs.
Reactions were performed in a total volume of 20 μL
(2 μL of cDNA, 2 μL of 10× PCR
Buffer, 3 μL of 2.5 mM dNTP, 1 μL of each primer, 0.5 μL of Taq
polymerase, and 10 μL of ddH2O). The whole genome of
BNoV strain Bo/CH/HB/BD/2019 was amplified by RT-PCR (oligonucleotide
primers are presented in Table 1). All PCR products were purified with
the PCR Purification Miniprep Kit (BIOMIGA, Shanghai, China) following
the manufacturer’s instructions. The PCR products were cloned into the
pMD19-T cloning vector (TaKaRa Biotechnology, Dalian, China). The
positive recombinant plasmids were verified by restriction enzyme
digestion and sequenced by Sangon Biotech (Beijing, China). The raw
genomic sequence fragments were imported to SeqMan in DNAStar (DNAStar,
Inc., Madison, WI, USA) for assembly and annotation. The complete BNoV
genome sequence has been deposited in GenBank (accession number:
MN480761). Sequencing and phylogenetic analysis of the complete genome
of the Bo/CH/HB/BD/2019 strain was performed using the Clustal W program
in DNA Star software. Then, 10,000 bootstrap replication was performed
using MEGA 7 software, and the phylogenetic tree was constructed by the
neighbor-joining method.