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.