2.4 PCR detection of PiCV
The PiCV was first detected using a PCR method targeting a 326-base
fragment of cap gene as 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 full genomes of
PiCV for 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 2×Master Mix (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 genomic
DNA, 25 μl Q5 High-Fidelity 2×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 5 min using an automated BioRad T100
Thermal Cycler (Bio-Rad Laboratories, Inc., CA, USA). The PCR products
(5 μl) were resolved on 1% (w/v) agarose gels, and followed by staining
with ethidium bromide. Finally, the bands of nucleic acid were
visualized with UV illumination inside a gel documentation apparatus
(Bio-Rad Laboratories, Inc., CA, USA) and saved as digital photographs.