2.4 Viral PCR identification
Total DNA was extracted from the culture supernatant using a DNA
Extraction Kit (TIANGEN BIOTECH (BEIJING)CO.,LTD.) according to the
manufacturer’s pro-tocol. DNA extracts were used in conventional PCR
assays. The sequences of the primers are FPV-F (5′-
GGATGGGTGGAAATCACAGC-3′) and FPV-R (5′-ATAACCAACCTCAGCTGGTC-3′) (Qiao
et al., 2001).The amplification was generally achieved by means of 35
cycles of predenaturation at 95˚C for 3min, denaturation at 95˚C for
30s, annealing at 50˚C-60℃ for 30 s and polymerization at 72˚C for 10
min. After electrophoresis in a 1.0% agarose gel and ethidium bromide
staining, the PCR products were excised from the gel and purified by a
commercial kit. For each amplified genome fragment, a pair of gene
specific primers are used for bidirectional sequencing (Sangon Biotech
Engineering (Shanghai) Co., Ltd.), and then the sequencing results are
spliced using DNAstar software to obtain a complete genome.