Establishment and application of the SYBR Green I real-time PCR assay for detection of porcine antiviral protein Mx1 gene

On the basis of the nucleotide sequences of porcine Mx1 gene(poMx1)available in GenBank,a pair of primers was designed and synthesized to amplify a specific 115 bp nucleotide fragment from Mx1 full-length gene.Then,the SYBR Green Ⅰ real-time PCR assay was established based on 10-fold dilution recombinant plasmid as the amplification template after it was cloned and sequenced.The results showed that the standard curve regression equation was Y=-2.986 3 lg X+38.239 3(R2=0.998 5),and that the standard curve was of high linearity,specificity,sensitivity and reproducibility.The production of poMx1 mRNA from virus-infected cells was quantitatively analyzed after swine kidney cells(PK-15)was stimulated by classical swine fever virus(HCLV strain).The data showed the production of poMx1 mRNA was much more after 4 h(P0.01),and then slowly reduced as time grew.So the results showed that this PCR assay could be applied to detect the expression level of poMx1 mRNA.