Abstract: In order to establish the assay of SYBR Green Ⅰ Real-time fluorescent quantitative RT-PCR for detecting three important swine cytokines IL-12, IFN-α and TNF-α.Three pairs of specific primers were designed according to the sequence of IL-12, IFN-α and TNF-α from GenBank to amplify the objective genes.The three genes were respectively cloned to the pMD18-T vector.The corresponding plasmids were identified by sequencing, and they were then used as quantitative template to construct the standard curve and to analyze the melting curve, detection sensitivity, specificity and repeatability.The results showed that Ct value of the three genes had good linear relationship (R²≥0.992) with the dilution ranging from 1×10¹ to 1×10⁶ copies/μL.The melting curve displayed a single peak and good repeatability.The established assay was used to detect the transcriptional level of IL-12, IFN-α and TNF-α mRNA in the swine PBMC inoculated PRRSV TJM-F92.The results indicated that mRNA levels of IL-12, IFN-α and TNF-α in the swine PBMC inoculated PRRSV TJM-F92 were all significantly increased (P< 0.01).The research provides the platform for quantitative analysis of swine cytokine IL-12, IFN-α and TNF-α.

Key words: swine; cytokines; SYBR Green Ⅰ; Real-time fluorescent quantitative RT-PCR