SYBR Green I-based RT-PCR Assay for Detecting IFN-β mRNA in Duck

  Objective  A method for detecting IFN-β in duck using SYBR Green I-based RT-PCR was developed.

  Methods  A pair of specific primers was designed according to the GenBank nucleotide sequence on IFN-β of duck (KT428159). The gene was cloned into a pET-30a vector, and the recombinant plasmid pET-30a-IFN-β severed to establish a standard curve. The specificity, sensitivity, and repeatability of the new methodology were determined.

  Result  The melting curves of measurement showed a sharp single peak at Tm=87.94±0.16 ℃ without non-specific amplification and primer dimers, indicating high specificity of the methodology. The Ct value ranged from 8.9 to 34.0 with a standard curve showing a linearity with R²＞99.5%. The detection limit on IFN-β was 2.84 copies/μL. The repeatability on the Ct data for the intra- and inter-groups had coefficients of variation below 0.13% and 1%, respectively.

  Conclusion  The newly developed assay was specific, sensitive, repeatable, and suitable for the quantitative detection of IFN-β mRNA in ducks.