Establishment of a real-time fluorescent quantitative PCR assay for detection of chicken IL-6, IL-17 and interferon-γ genes

【Objective】A real-time PCR method for detection of chicken interleukin-6(IL-6), interleukin-17(IL-17) and interferon-γ(IFN-γ) mRNA was established to provide references for quantitative detection of cytokines and virus pathogenic mechanism. 【Method】Four specific pairs were designed according to the chicken's IL-6, IL-17, IFN-γ and 3- glyceraldehyde phosphate dehydrogenase(GAPDH) gene available sequences in GenBank. The positive recombinant plasmids which were constructed from cDNA of chicken embryo fibroblast were used for the development of standard curve of SYBR GreenⅠreal-time PCR. The conditions were optimized such as the annealing temperature, and the concentration of primer. The specificity, sensitivity, and repeatability were tested. 【Result】The results showed that each gene melting curve had a single peak. Each gene amplification efficiency was 98.2%, 99.2%, 102.0% and 100.8%,and corresponoling Standard error was 0.00666,0.00813,0.00365 and 0.00458. Sensitivity tests results showed that lower limit of detection for each gene was 100 DNA copy number. The repeatability tests results showed that the coefficient of variation within the group was less than 2.00%. 【Conclusion】The established SYBR GreenⅠreal-time PCR assay was specific, sensitive and repeatable, which provided a rapid and quantitative detection method for chicken IL-6, IL-17 and IFN-γ mRNA transcription.