2.2 PCR detection
Viral DNA was extracted from 200 μL supernatants using DNA Miniprep Kit (Omega, Norcross, Georgia, USA) according to the manufacturer’s instructions, and used for PRV detection by polymerase chain reaction (PCR). A pair of primers gEp-L/R (gEp-L: 5’-TGGGACACGTTCGACCTGATG-3’, gEp-R: 5’-CCTT GATGACCGTGACGTACT-3’) were designed to amplify partial gE gene. PCR was performed in a 25 μL volume mixture consisting of 12.5 μL Premix Taq (Takara, Dalian, China), 3 μL the extracted DNA template, 7.5 μL of ddH2O, 1 μL DMSO and 0.5 μL each of primer (50 μM), and the cycling protocol was an initial denaturation at 95 °C for 5 min, followed by 35 cycles at 95 °C for 30 s, 53 °C for 30 s, and 72 °C for 1 min, with a final step of 72 °C for 10 min. PCR product was visualized by electrophoresis in a 1.5 % agarose gel containing ethidium bromide under ultraviolet light.
2.3 Virus isolation
For virus isolation, homogenate supernatants of positive tissue samples were filtered using a 0.22 µm filter (EMD Millipore, Billerica, MA, USA), and inoculated into ST cells. After cytopathic effect (CPE) appeared, cell cultures were collected and further used to 3 cycles of plaque purification, and confirmed by PCR with primers gEp-F/R. Subsequently, plaque fluid was inoculated into ST cells and cultured to 7th generation. The 50% tissue culture infectious dose (TCID50) were determined for PRV isolates, and calculated by Spearman-Karber method.