鸭IFN-β mRNA SYBR Green Ⅰ荧光定量RT-PCR检测方法的建立

方铁辉; 董慧; 肖世峰; 王劭; 程晓霞; 林锋强; 朱小丽; 陈秀琴; 郑敏; 陈仕龙; 陈少莺

doi:10.19303/j.issn.1008-0384.2021.10.004

鸭IFN-β mRNA SYBR Green Ⅰ荧光定量RT-PCR检测方法的建立

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

摘要

摘要:
目的建立一种检测鸭IFN-β mRNA转录水平的SYBR Green Ⅰ实时荧光定量RT-PCR检测方法。
方法根据GenBank中鸭IFN-β（KT428159）核苷酸序列设计并合成特异性引物，将鸭IFN-β基因克隆至pET-30a载体，以此构建的pET-30a-IFN-β阳性重组质粒作为阳性标准品，采用SYBR Green Ⅰ实时荧光定量PCR检测，构建标准曲线，并进行引物特异性、灵敏度及重复性试验。
结果该扩增特异性强，无引物二聚体及非特异性产物，熔解曲线单峰（Tm=87.94±0.16 ℃）；Ct值在8.9～34.0线性拟合程度高，相关系数R²＞99.5%；灵敏度高，最低检测限为2.84 copies·μL⁻¹；重复性好，对来自临床的3种组织样品检测的组内变异系数小于0.13%，组间变异系数不超过1%。
结论该方法特异性强、灵敏度高、重复性好，为鸭IFN-β mRNA表达水平的定量分析提供了技术手段。

Abstract:
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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