Abstract: The assay was aimed to establish a SYBR Green Ⅰ fluorescent quantitative RT-PCR method to detect the mRNA of type Ⅰ interferon effect factor ISG15, ISG56, Mx1, OAS and PKR in HeLa cells. Using this method, foot and mouth disease virus (FMDV) L protein inhibiting the type Ⅰ IFN signaling pathway was evaluated. Total RNA was extracted by TRIzol reagent and Oligo d(T)₁₅ was used for reverse transcription, the target gene was amplified individually by PCR and cloned into pMD18-T vector, the positive recombinant plasmid was constructed and used as template for SYBR Green Ⅰ fluorescence quantitative RT-PCR. The standard curve and melting curve were established and the sensitivity, specificity and reproducibility assay were conducted. Using this established method, the inhibiting effect on type Ⅰ IFN effect factor of FMDV L protein was detected. The ISG15, ISG56, Mx1, OAS the PKR relative mRNA level of HeLa cells transfected with eukaryotic expression plasmid expressing FMDV L protein were obviously reduced compare to HeLa cells transfected with empty vector or eukaryotic expression plasmid expressing GST. The SYBR Green Ⅰ fluorescent quantitative RT-PCR method was established, which laid the foundation to evaluate the mRNA levels of type Ⅰ IFN effect factor in HeLa cells.This method was successfully applied to FMDV L protein inhibition of type Ⅰ IFN signaling pathway.

Key words: Real-time fluorescent quantitative RT-PCR; SYBR GreenⅠ; foot and mouth disease virus; L protein; interferon