To amplify the newly synthesized rearranged PCV3 gene, PCV3 M1, PCV3 L2 primers, and KFX-101 high fidelity enzymes (TOYOBO, Japan) were adopted. The final volume of reaction fluid reached 100 μL. The PCR cycling conditions included: pre-denaturation at 94°C for 2 min, 30 cycles at 98°C for 10 s, 58°C for 30 s, and 68°C for 2 min, as well as the final extension for 10 min at 68°C. PCR products were preserved at 16°C for subsequent experiments.
The amplified PCR products of rearranged PCV3 gene were purified following the instructions of the DNA Purification Kit (Takara Bio, Dalian, China). Subsequently, the purified PCR product was digested by HindIII restriction endonuclease (NEB, Beijing, China) and then incubated in a water bath at 37°C for 5 h. Then, the digested products of HindIII restriction endonuclease were purified following the manufacturer’s instructions (Takara Bio, Dalian, China). Next, the purified DNA fragments were connected based on T4 DNA ligase instructions (NEB, Beijing, China) at 16°C for 12 h. Last, the cyclized PCV3 DNA was harvested.
The amplified PCR products of rearranged PCV3 gene were purified following the instructions of the DNA Purification Kit (Takara Bio, Dalian, China). Subsequently, the purified PCR product was digested by HindIII restriction endonuclease (NEB, Beijing, China) and then incubated in a water bath at 37°C for 5 h. Then, the digested products of HindIII restriction endonuclease were purified following the manufacturer’s instructions (Takara Bio, Dalian, China). Next, the purified DNA fragments were connected based on T4 DNA ligase instructions (NEB, Beijing, China) at 16°C for 12 h. Last, the cyclized PCV3 DNA was harvested.