Abstract: In order to develop a SYBR GreenⅠ Real-time PCR assay for detection of chicken IL-1β,IL-18 and tumor necrosis factor-α (TNF-α) genes, four specific primer pairs were designed according to the chicken's IL-1β,IL-18, TNF-α and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene sequences in GenBank. The four fragments were amplified by RT-PCR from chicken embryo fibroblasts, 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 GreenⅠfor detection of chicken IL-1β,IL-18, TNF-α and GAPDH were established. The results showed that each gene's melting curve also had a single peak,each gene's amplification efficiency was 101.2%, 95.6%, 100.1% and 98.2%, R² was 0.9996,0.9998,0.9957 and 0.9989. Moreover,the assays were highly sensitive,the detection limit of 100 copies in 35 Ct and each gene's coefficient of variation less than 1.4 percent for intra-assay. This reliable Real-time PCR assay might be used for decting chicken's IL-1β,IL-18 and TNF-α mRNA expressing and provided the basis for quantitative analysis of cytokine expression in host cell after virus infect which cause immunosuppressive diseases.

Key words: chicken; IL-1β; IL-18; TNF-α; Real-time PCR