Development of a Real-time PCR Assay Based on SYBR Green I for Detection of Porcine IL-2,IL-12p40 and IFN-γ

A pair of primers was designed according to the sequence of porcine Th1-type cytokines,IL-2,IL-12p40 and IFN-γ gene as well as housekeeping gene β-actin,respectively,and a recombinant plasmid containing the target gene was constructed as a standard control for each cytokine.Then,a real-time PCR assay based on SYBR c for detection of IL-2,IL-12p40,IFN-γ and β-actin was established,respectively.Every assay had a good linear relationship between initial templates and Ct values,and the correlation coefficient of the standard curve was over 0.997.It was highly sensitive and had a detection limit of 100 copies/μL.It was highly specific and there was single specific melting peak of every cytokine.It was highly reproducible and had a coefficient of variation less than 3.5 percent for both intra-and inter-assay.The established assays were successfully used to detect IL-2,IL-12p40 and IFN-γ mRNA expression levels in peripheral blood mononuclear cells from piglets experimentally infected with porcine reproductive and respiratory syndrome virus(PRRSV).The high sensitivity,specificity and reproducibility of the assays indicated that the SYBR Green Ⅰ real-time PCR could be used as an effective tool for detection and quantification of these Th1-type cytokines.