Establishment and application of a real-time fluo rescent quantitative RT-PCR assay for detection of bovine CD80 and CD86 genes

To establish a SYBR Green real-time quantitative PCR assay for detecting bovin e co-stimulates genes CD80 and CD86,three pairs of primers were designed accord ing to the bovine(BoCD80,CD86)gene sequences available in GenBank,while the bo vine glyceraldehyde-3-phosphate dehydrogenase(BoGAPDH)gene was used as an int erna1 control.The gene was amplified by the traditional PCR.The PCR product was inserted into pMD18-T vector and sequenced.The positive recombinant plasmid was used as quantitative template to generate s tandard curve and melting curve.Sensitivity,reproducibility and specificity of t he assay were determined.The results showed that the Ct of BoCD80,CD86 or BoGAPDH genes had a good linear relationship(r20.991)with the standard samples from 1×102 to 1×108 copies/μL,and the melting curve showed a single peak and more repr oductive and specific.The established real-time PCR assay could quickly detect clinical specimens.