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).