Development of a TaqMan real-time RT-PCR assay for detecting porcine IL-2, IL-4, IL-10, and IFN gamma

To develope a TaqMan real-time RT-PCR assay for detecting porcine IL-2, IL-4, IL-10, and IFN-γ, the conserved regions of porcine IL-2, IL-4, IL-10, and IFN-γ genes were amplified with Wuzhishan miniature pig cDNA as the template and cloned into pMD18-T to construct the standard recombinant plasmids for establishing calibration curve, respectively. The results showed that the real-time RT-PCR assay was sensitivity and repeatability. The detection limit of IL-4 mRNA was 1 copies/μL, while the detction limit of IL-2, IL-10, and IFN-γ was 102 copies/μL, respectively. The coefficient of variation for both intra-assay and inter-assay was lower than 5%. The evaluation results further demonstrated that the established real-time RT-PCR detection was reliable. This method could be used to evaluate the immune response in pigs.