Development of SYBR GreenI-based real-time PCR assays for detection of porcine IL-1β and IL-18

The specific primers were designed and synthesized according to the nucleotide sequence of the porcine pro-inflammatory cytokines,i.e.IL-1β,IL-18 genes as well as housekeeping gene β-actin available in GenBank.IL-1β and IL-18 play important roles in mediating inflammatory response.The three fragments were amplified by RT-PCR from 3D4 porcine alveolar macrophages,cloned,and sequenced.The recombinant plasmids containing the target gene were constructed,and used as the real-time PCR standard templates.Real-time PCR assays based on SYBR Green Ⅰ for detection of IL-1β,IL-18 and β-actin were established.The results showed a good linear relationship between template copy number and circulation number,and the correlation coefficients(R2) of the standard curves for IL-1β,IL-18 and β-actin were over 0.990.Also,these assays were highly specific and there was single specific melting peak for every cytokine.Moreover,the assays were highly sensitive and had a detection limit of 1.0×102copies in 1 μl of initial templates.Finally,it was highly repeatable and had a coefficient of variation less than 4 percent for intra-assay.