Development and application of a SYBR Green I-based real-time PCR assay for detection of porcine IL-4, IL-6 and IL-10.

Real-time PCR assays based on SYBR GreenⅠfor detection of IL-4, IL-6, IL-10 and β-actin were established using primers derived from porcine Th2-type cytokines (IL-4, IL-6 and IL-10) gene. The assays were highly sensitive and had a detection limit of 1×101 copies/μL of initial templates. These assays were highly specific and there was single specific melting peak for every cytokine. It was highly reproducible and had a coefficient of variation less than 3 percent for both intra-and inter-assay. The established assays were successfully used to detect IL-4, IL-6 and IL-10 mRNA expression levels in peripheral blood mononuclear cells (PBMCs) in piglets experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV). The high sensitivity, specificity and reproducibility of the assays indicated that the SYBR GreenⅠreal-time PCR could be used as an effective tool for detection and quantification of Th2-type cytokines.