Establishment and application of a real-time fluorescent quantitative RT-PCR assay for detection of bovine Th1/Th2 cytokines

To establish a SYBR-Green Ⅰ real-time quantitative PCR assay for detection of bovine's Th1/Th2 cytokines.According to the bovine's Th1/Th2(BoTNF-α,BoIFN-γ,BoIL-2,BoIL-4,BoIL-6,BoIL-8 and BoIL-10) cytokines gene sequences available in GenBank,eight pairs of primers were designed for developing a SYBR Green Ⅰ quantitative real-time PCR method for detection of Th1/Th2 cytokines of bovine while the bovine glyceraldehyde-3-phosphate dehydrogenase(BoGAPDH)gene was used as an interna1 control.The genes was amplified with traditional PCR.The PCR product was served pMD18-T vector and sequenced,The positive recombinant plasmid was used as quantitative template to generate standard curve and melt curve.Sensitivity,reproducibility and specificity assays were determined.The results showed that the Ct of Th1/Th2 cytokines had a good linear relationship(r20.991) with the standard samples from l×101 to 1×107 copies/μL,and the melting curve showed a single peak and more reproductire and specific.The established real-time PCR assay could quickly detect Th1/Th2 cytokines genes in expansion range with high efficiency,thus providing the basis of quantitative analysis of Th1/Th2 cytokines gene expression.