Establishment of a SYBR Green I real-time quantitative RT-PCR assay for detection of bovine alpha-interferon, bata-interferon and gamma-interferon mRNA.

This study aimed to develop a SYBR Green Ⅰ real-time quantitative RT-PCR assay for rapid detection of bovine IFN-α,IFN-β and IFN-γ(BoIFN-α,-β and-γ) mRNA.The assay was established using four pairs of specific primers designed according to the BoIFN-α,-β and-γ gene sequences,with the bovine glyceraldehyde-3-phosphate dehydrogenase(BoGAPDH) mRNA as an internal control.The target genes were amplified by conventioanl RT-PCR,and used as templates for generating standard curve and melt curve.The results showed that the Ct value of BoIFN-α,-β,-γ and BoGAPDH mRNA had a good linear relationship(r20.991) with the standard from 10 copies/μL to 107 copies/μL,and the melting curve showed a single peak.The reproducibility,specificity and sensitivity of the assay were also higher than conventional RT-PCR in detecting respective target mRNA.Therefore,the real-time quantitative PCR assay could be effective method for quantitative analysis of IFN-α,-β and-γ gene expression.