Establishment of a competitive RT-PCR assay for quantitative detection of porcine IFN-γ mRNA

The total RNA was extracted from aseptically-isolated porcine peripheral blood mononuclear cells(PBMC),which were cultured with induction in vitro of ConA for 15h.From the total RNA,an IFN-γ gene of 435bp was amplified by RT-PCR.The recombinant plasmid pMD-IFN435 was constructed by ligating the amplified IFN-γ gene into the vector pMD18-T.A gene fragment of 257bp,with a PstⅠ digestion site,was amplified from pMD-IFN435 with another pair of primers by PCR.The recombinant plasmid pMD-IFN257 was constructed by ligating the 257bp gene fragment into the vector pMD18-T.The recombinant plasmid pMD-IFN367 containing 367bp of the target gene was constructed after the gene fragment from pMD-IFN257 digested by PstⅠ+SalⅠ was ligated with the gene fragment from pMD-IFN435 digested by Pst+SalⅠ.The linear regression equation of the standard competitive curve(y=0.414x-1.864) for the accurate and convenient detection of porcine IFN-mRNA was developed by using pMD-IFN367 and pMD-IFN435 as PCR competitive templates.