Development and application of SYBR Green I real-time PCR assays for detection of sheep IFN-γ and IL-2 genes.

Real-time PCR assays based on SYBR GreenⅠfor detection of IFN-γ,IL-2 and GAPDH genes were established using primers derived from sheep Th1-type cytokine(IFN-γ,IL-2) genes.The assays were highly sensitive and had a detection limit of 1×10 3copies/μL of initial templates.These assays were highly specific and there was single specific melting peak for each gene.They were highly reproducible and had a coefficient of variation less than 3 percent for intra-and inter-assays.The established assays were successfully used to detect IFN-γ and IL-2 mRNA expression levels in peripheral blood leukocytes in lamb experimentally infected with orf virus.The high sensitivity,specificity and good reproducibility indicated that the developed SYBR Green Ⅰreal-time PCR assays were effective tools for the detection and quantification of sheep Th1-type cytokines.