2.2. Construction of standard plasmids for quantitative PCR
Genomic sequences of PBoVs were downloaded from the GenBank and aligned
using the MegAlign program of DNAStar software (version 7.1, DNASTAR
Inc., Madison, USA). The highly conserved sequences within theNS1 gene were determined from an alignment of nine PBoV1/2
genomic sequences (GenBank accession number HM053693, HM053694,
HQ291309, KF025392, KF025393, KF206155, KF206157, KF206161 and
KM402139), and a pair of primers PBoV1/2-F/R was designed using the
Primer Premier (version 6.0, USA) software based on the reference
sequence (HM053693) (Table S1). The specific primers for PBoV3/4/5 were
found from an alignment of nine sequences of PBoV3/4/5 (GenBank
accession number JF429834, JF713714, KC473563, JF713715, JN681175,
JF429836, KF206158, JN621325 and JN831651), and a pair of primers
PBoV3/4/5-F/R targeting the VP 1 gene of PBoV3/4/5 was designed
based on the reference sequence (JF429834) (Table S1).
PCR amplifications with primers PBoV1/2-F/R and PBoV3/4/5-F/R were
performed using the PCT-200 Peltier thermal cycler (MJ Research, USA).
The positive PCR products were purified using the Gel Extraction Mini
Kit (Watson Biotechnologies. INC) according to the manufacturer’s
instructions, and cloned into pMD18-T vector (Takara, Dalian, China) to
produce the recombinant plasmids, which were designated as pMD18-327 and
pMD18-209 respectively. Plasmids were transformed into Escherichia
coli DH-5α competent cells (Takara) for sequencing, and their
concentrations and purities were quantified by ultraviolet absorbance at
260 nm and 280 nm using a Nano-100 micro-spectrophotometer (Thermo
Fisher Scientific, Waltham, MA, USA). The copy numbers of plasmids were
determined and 10-fold serial diluted in TE buffer (10mM Tris–HCl, 1mM
EDTA).