2.3. Complete genome amplification and sequencing
We amplified the complete PCV4 genome from the positive samples. For the
genomic sequence analysis, two pairs of primers (first forward,
5’-CCGTGAGTTCCCGTCTGTAT-3’, located at nt 464-483; first reverse,
5’-TGGAGACTATGTGGGGGAAG-3’, located at nt 1355-1374; second forward,
5’-GCTGGAAGTGGAGGGTGTG-3’, located at nt 1220-1238; second reverse,
5’-CCCCTTCTCTTGTACATGTCT-3’, located at nt 600-620) were designed in
this study. The designed primer sequences are based on the genomic
sequence of PCV4 strain HNU-AHG1-2019 (accession number: MK986820). The
PCR thermal profile was initially denatured for 5 min at 95℃, followed
by 40 cycles of 30 sec at 95℃, 2 min at 55℃, 1 min 20 sec at 72℃ for,
and final extension for 7 min at 72℃. After screening, positive samples
were sent to a commercial facility (Macrogen Co., Ltd. Korea) for
sequencing.