2.4 PCR detection of PiCV
The PiCV was first detected using a PCR method targeting a 326-base fragment of cap gene described by Freick et al. (2008). The primer sequences were: PiCV-s, 5’-TTGAAAGGTTTTCAGCCTGGC-3’ and PiCV-as, 5’-AGGAGACGAAGGACACGCCTC-3’ (Freick et al., 2008). The PiCV full genomes of all the positive samples were amplified by PCR using the primers: PiCV-1F, 5’-ACCCGCGACTTGGAGCCACGGAG-3’ and PiCV-1R, 5’-TTCGCTCCCGCATTCGCGGTCGCT-3’, PiCV-2F, 5’-GACACTAGTAAAGGGACCCAAGCCA-3’ and PiCV-2R, 5’-AAGCCTTGCAGATGCGGGGT-3’, respectively. PCR was performed by using Q5 Hot Start High-Fidelity 2X Master Mix (NEB, MA, USA) containing NEB Q5 Hot Start High- Fidelity DNA Polymerase (NEB, MA, USA). The contents of the three reactions mixture in a 50 μl reaction volume were as follows: 0.5 μM forward primer, 0.5 μM reverse primer, 1 μg of genomic DNA, 25 μl of Q5 High-Fidelity 2X Master Mix (NEB, MA, USA) and an appropriate volume of Nuclease-Free Water. The cycling parameters were 30 cycles of 98°C for 10 s, 55°C for 30 s and 72°C for 30 s, followed by a final extension at 72°C for 72 min using an automated BioRad T100 Thermal Cycler (Bio-Rad Laboratories, Inc., CA, USA). PCR products (5 μl) were resolved on 1% (w/v) agarose gels, stained with ethidium bromide, visualized with UV illumination inside a gel documentation apparatus (Bio-Rad Laboratories, Inc., CA, USA) and saved as digital photographs.