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.