2.2. The detection of PCV4
The detection of PCV4 was based on PCR assay utilizing specific primers designed in the previous study (Tian et al., 2020). The upstream primer (PCV4F) was 5’-GTTTTTCCCTTCCCCCACATAG-3’, located at nucleotide (nt) 1347-1368, and the downstream primer (PCV4R) was 5’-ACAGATGCCAATCAGATCTAGGTAC-3’, located at nt 1713-1737. All primers used in this study were synthesized by Macrogen Co., Ltd., Korea.
The PCR reaction mixture consisted of 2µL of template DNA, 1µL of each primer (10µM) and 16µL of Master mix solution (iNtRON Biotechnology, Inc. Korea). The PCR thermal profile was an initial denaturation for 5 min at 95℃, followed by 40 cycles of 95℃ for 30 sec, 58℃ for 30 sec, 72℃ for 30 sec, and a final extension for 7 min at 72℃. The PCR products were analyzed through electrophoresis on 1% agarose gel with Red Safe DNA nucleic acid staining solution (iNtRON Biotechnology, Inc. Korea).