Establishment of a Real-time Fluorescent Quantitative RT-PCR Assay for Detection of Porcine IFN-β and IRF-3 genes

To establish real-time fluorescent quantitative RT-PCR assays for detecting porcine IFN-β and IRF-3,sevral specific primer pairs were designed according to the porcine's IFN-β and IRF-3 gene sequences available in GenBank,and the porcine β-actin gene was used as an internal gene control.The total RNA was extracted from porcine alveolar macrophages.The reverse transcriptase PCR was used to obtain the first strand cDNA.Fragments for IFN-β and IRF-3 were amplified by PCR from the synthesized cDNA using the designed specific primers.The PCR products were purified and cloned into pMD-19T vector.The positive recombined plasmids were serially diluted and used as a standard.The standard and melting curve was analyzed.The results showed that the Ct value of β-actin,IFN-β and IRF-3 genes had a good linear relationship(R2≥0.997) and the melting curve showed a single peak.The established real-time PCR methods can detect 100 copies of IFN-β and IRF-3 mRNA.The developed real-time PCR using SYBR GreenⅠdye had high sensitivity,sepcifity and reproductivity,and could used as an effective tool for detection and quantification of IFN-β and IRF-3.