The linear ssDNA was also used for the DNA binding assay. The preparation of linear ssDNA followed the protocol described in Marimuthu et al.  using the biotin-streptavidin separation method. First, a biotinylated DNA fragment containing the whole CAV genome was amplified by PCR using the EmeraldAmp Max PCR Master kit (Takara, Japan) from pCAV with a designed primer set, including the reverse biotinylated primer Biotin-CAV-r: biotin-labelled-GATTGT GCGGTGAACGAATTAG, and the forward regular primer CAV-f: GAATTCCGAGTGGTTACTATTC. After PCR amplification, the biotinylated PCR product was then immobilized on 40 μl Dynabeads M-280 Streptavidin magnetic beads (Invitrogen, USA) and incubated at 4 °C overnight. After washing the DNA-bonded beads twice with B/W buffer (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 M NaCl), the washed beads were incubated in 150 μl elution buffer (0.1 M NaOH, 1 mM EDTA, pH 13.0) to perform alkaline denaturation. Under the high alkaline environment, the desired non-biotinylated strand can be separated from the biotinylated strand and suspended in the supernatant. After magnet adsorption, the supernatants were collected, and the linear ssDNA was further purified by a PCR clean-up kit (Geneaid, Taiwan). The linear ssDNA was diluted to 50 ng/ml with DNA-binding buffer and store at − 20 °C until required.