SYBR Green I-based RT-PCR Assay for Detecting IFN-β mRNA in Duck
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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。 -
关键词:
- 鸭 /
- IFN-β /
- 荧光定量RT-PCR /
- SYBR Green I /
- mRNA
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. -
Key words:
- Duck /
- IFN-β /
- qRT-PCR /
- SYBR Green I /
- mRNA
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图 1 鸭IFN-β全基因的PCR扩增及重组质粒的构建
注：A，鸭脾脏IFN-β基因的扩增；B，重组质粒的IFN-β基因扩增；C，重组质粒的双酶切鉴定。
Figure 1. PCR amplification of duck IFN-β and construction of recombinant plasmid
Note: A: amplification of IFN-β from duck spleen; B: IFN-β amplification from recombinant plasmid; C: identification of recombinant plasmid by double restriction enzyme digestion.
图 2 不同组织IFN-β的常规RT-PCR和荧光定量RT-PCR
注：1～4分别为脾脏、肝脏、胰腺及DEFs细胞的常规PCR扩增；M为DL 2000 DNA Marker；5～8分别为脾脏、肝脏、胰腺及DEFs细胞的qPCR扩增；9、DEPC水。
Figure 2. Conventional RT-PCR and qRT-PCR amplification of IFN-β in different tissues
Note: 1–4: conventional PCR amplification of IFN-β in spleen, liver, pancreas, and DEFs cells, respectively; M: DL 2000 DNA marker; 5–8: qPCR amplification of IFN-β in spleen, liver, pancreas, and DEFs cells, respectively; 9: DEPC water.
图 3 鸭4种组织（肝、脾、胰和DEFs）IFN-β 荧光定量RT-PCR熔解曲线
Figure 3. qRT-PCR melting curve of duck IFN-β
图 4 IFN-β 荧光定量RT-PCR扩增曲线
注：1～8分别为2.84×108、2.84×107、2.84×106、2.84×105、2.84×104、2.84×103、2.84×102、2.84×101 copies·μL−1；9、DEPC水。
Figure 4. Amplification curve of qRT-PCR for IFN-β
Note: 1–8: 2.84×108, 2.84×107, 2.84×106, 2.84×105, 2.84×104, 2.84×103, 2.84×102, and 2.84×101 copies·μL−1, respectively. 9: DEPC water.
表 1 引物核苷酸序列及扩增片段大小
Table 1. Primer sequences and amplified fragment sizes
引物
Primers核苷酸序列
Nucleotide sequence（5′-3′）扩增片段大小
Amplified fragment size/bpFull-IFN-β-F CGCGGATCCATGCCTGGGCCATCAGC 729 Full-IFN-β-R CCCAAGCTTTCACGCCGTGGGCTTGT qIFN-β-F GGGCTCCGCAACCTTCACC 165 qIFN-β-R TGCTTGGCTCTTCATCCGCCGTA 注：下划线区域为酶切位点序列。
Note：The underlined parts are the sequences of restriction sites.
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表 2 实时荧光定量RT-PCR敏感性检测
Table 2. Sensitivity of qRT-PCR assay
质粒拷贝数
Copy number
/（Copies·μL−1）2.84×103 2.84×102 2.84×101 2.84×100 2.84×10−1 Ct值（平均值±标准差）
Ct （Means±SD）26.38±0.03 29.87±0.10 33.98±0.05 36.13±1.12 − 变异系数 CV/% 0.11% 0.33% 0.15% 3.10% −
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表 3 实时荧光定量PCR方法的批内与批间重复性评价结果
Table 3. Reproducibility of intra- and inter-qRT-PCR assays
组织
Tissue组内重复性试验Ct值
The Ct values of intra-assay组间重复性试验Ct值
The Ct values of inter-assay平均值±标准差
Means±SD变异系数
CV/%平均值±标准差
Means±SD变异系数
CV/%脾 Spleen 26.13±0.03 0.11 26.37±0.21 0.80 肝 Liver 32.63±0.04 0.12 32.41±0.20 0.62 胰 Pancreas 31.51±0.04 0.13 31.54±0.30 0.95
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