2.4 Complete genomic sequence amplification
Another three pairs of specific primers (Q1F/Q1R, Q2F/Q2R and Q3F/Q3R)
were designed according to the reference sequence (HM053693) and used to
amplify three overlapping fragments covering the entire PBoV genome
(Table S1). For the two primer sets (Q1F/Q1R and Q2F/Q2R), PCR was
carried out in 25 μL of the reaction mixture: 12.5 μL of 2Phanta Max
Buffer, 2 μL of sample DNA, 1 μL of Phanta Max Super, 0.5 μL of dNTP
Mix, 0.5 μL of each primer (50 μM), and 8 μL of ddH2O.
The PCR parameters were as follows: pre-denaturation at 94°C for 3 min;
followed by 35 cycles of denaturation at 98°C for 10 s, annealing at
55°C for 15 s, extension at 72°C for 2 min, and then followed by a final
extension step at 72°C for 10 min. For the Q3F/Q3R primer sets, the PCR
reaction mixture consisted of 12.5 μL of 2Es Taq Master Mix (Dye), 3 μL
of sample DNA, 0.5 μL of each primer (50 μM) and ddH2O
to a total volume of 25 μL. The reaction was performed by preheating for
5 min at 95°C, followed by 35 cycles of 95°C for 45 s, 60°C for 45 s,
and 72°C for 2 min, and a final extension step for 10 min at 72°C. The
purified
products were inserted into the pMD18-T Vector (Takara) for sequencing,
and all sequencing reactions were performed in duplicate.