Establishment of the TaqMan real-time RT-PCR for detecting IFN-γ, IL-2, and TNF-α from pig whole blood and assessment of cellular immune response in pigs vaccinated with classical swine fever vaccine.

For detection of porcine cytokines from pig whole blood,the real time TaqMan RT-PCRs were developed using specific primers and fluorescent probe designed according to the genes of IFN-γ,IL-2 and TNF-α in GenBank.The results showed that the established method had a linear dynamic range from 101 copies/μL to 109 copies/μL,with the correlation coefficient R2 of 0.999.The assay was used to assess cellular immune response of classical swine fever(CSF) attenuated live vaccine in pigs,the results demonstrated that the IFN-γ,IL-2 and TNF-α mRNA expressions were significantly increased from 3 to 10 days(p0.05), respectively;However,the higher expression of IFN-γ lasted to 14 days when CSFV stimulated cells in comparing with unstimulated control cells in pigs post immunization with CSFV attenuated vaccine.Nevertheless,those mRNA expressions had no obvious difference between CSFV stimulated cells and control cells in unvaccinated pig group.The results suggested that the method was highly specific,sensitive and reproducible which was reliable for assessment cellular immune response for vaccine immunizations.