• 中文核心期刊
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  • 中国科技核心期刊
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FANG T F, DONG H, XIAO S F, et al. SYBR Green I-based RT-PCR Assay for Detecting IFN-β mRNA in Duck [J]. Fujian Journal of Agricultural Sciences,2021,36(10):1139−1144. DOI: 10.19303/j.issn.1008-0384.2021.10.004
Citation: FANG T F, DONG H, XIAO S F, et al. SYBR Green I-based RT-PCR Assay for Detecting IFN-β mRNA in Duck [J]. Fujian Journal of Agricultural Sciences,2021,36(10):1139−1144. DOI: 10.19303/j.issn.1008-0384.2021.10.004

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.
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