2.2 DNA isolation and polymerase chain reaction (PCR)
The viral genome was extracted from swine serum or tissue using the
EasyPure Viral DNA/RNA Kit (TransGen Biotech, Beijing, China) according
to the manufacturer’s instructions. Two primer pairs were designed based
on the reference sequences of the PCV4/HNU-AHG1-2019 strain (NO.
MK986820.1), and published primers and protocols were used to detect
PCV4 (Table S1). The PCR mixture contained 2 μL of extracted DNA, 2 μL
of primer pairs (10 μM), 25 μL of 2×Phanta Max Master Mix (Vazyme,
Nanjing, China), and 21 μL of DNase/RNase-Free water. The PCR
amplification conditions were as follows: predenaturation for 3 min at
95 °C; followed by 35 cycles of 15 s at 95 °C, 15 s at 62 °C, extension
for 1 min at 72 °C; and a final extension for 5 min at 72 °C.
Subsequently, the PCR products were separated using 1.2% agarose gel
electrophoresis of DNA and cloned into a pMD18-T vector (Takara Co.
Dalian). The recombinant vectors were amplified in Escherichia
coli (E. coli , DH5α) for sequencing.