Development of TaqMan fluorescent quantitative RT-PCR assay for detection of porcine IFN-γ mRNA.

To establish a fluorescent quantitative RT-PCR assay for detection of porcine interferon-γ(IFN-γ),specific primers and fluorescent probes based on porcine IFN-γ gene and housekeeping gene cyclophilin A(CyPA) were designed,respectively.Using the plasmids pMD18-T-IFN-γ and pMD18-T-CyPA as standard products,a real-time quantitative TaqMan reverse transcription-polymerase chain reaction(RT-PCR) was performed to construct the standard curves of porcine IFN-γ and CyPA.The results indicated that the assay for the quantitation of porcine IFN-γ template worked well from 1×101 to 1×107 copies/μL with excellent linearity,the coefficient correlation r2 reached 0.999 and amplification efficiency was higher than 99.0%.The sensitivity was sufficient to detect the least template of 100 copies.The established porcine IFN-γ specific real-time RT-PCR was time-saving,highly sensitive,specific,reliably reproducible and suitable to detect clinical specimens.