Establishment and application of a quantitative real-time PCR assay for detection of chickens' cytokines.

The specific primers were designed according to the conserved sequences of IL-6,IL-1β,IFN-γ,and TGF-β4 mRNAs,respectively.Total RNA was extracted from chicken macrophage cell HD11 stimulated by flagellin,then it was reverse-transcribed into cDNA.A quantitative real-time PCR(QRT-PCR) method was established to evaluate the relative expression levels of the four cytokines,respectively.There was a single peak in the melting curve,and the amplification curves of the same cDNA were superpositions in teraplicate.Stimulating by flagellin,the relative expression levels of inflammatory IL-6 and IL-1β were 14.90 and 29.09 in HD11 cells respectively and were higher than that of the controls(P0.05),while the relative expression level of IFN-γ and anti-inflammatory TGF-β4 didn't change significantly in HD11 cells(P0.05).The expression levels of inflammatory IL-6 and IL-1β were detected to be 6.19 and 2.39 respectively,in three chickens' heterophils infected by Salmonella by this method and were higher than that of the controls(P0.05),while the relative expression level of TGF-β4 didn't change significantly(P0.05).The results showed that the established QRT-PCR was specific and reproducible,and could be used for the rapid and accurate detection of chicken cytokines.