Establishment of a Real-time Fluorescence Quantitative PCR Assay for Detection of Duck RIG-I Like Receptors and interferon Genes

This study established detection of duck RIG-I,MDA5,IFN-αand IFN-γ mRNA relative expression levels synchronous of SYBR GreenⅠreal-time quantitative PCR for the first time.GAPDH was used as an internal reference.Cloning plasmids of objective genes were used as the standards for establishing standard curves for analysis of the melting curve,repeatability and stability.The linear range of Ct values of the method was 12.0to32.0.The amplification efficiencies of target genes(RIG-I,MDA5,IFN-α,IFN-γ)and the reference gene(GAPDH)were from 95% to 105%,and the correlation coefficients were above 0.99.The melting curve of amplification product only appeared a specific single peak,and there was no primer dimer or other non-specific product formation.Tm values of RIG-I,MDA5,IFN-α,IFN-γand GAPDH were(81.9±0.33),(81.9±0.32),(93.6±0.23),(81.8±0.28)and(87.8±0.24)℃,respectively.The sensitivity reached 100copies·μL-1,and repeatability and stability were good.This method would lay the foundation for further study of correlation between duck RIG-I like receptor,interferonand immune function from the mRNA levels.