| 韩剑1, 罗明1*, 徐金虹2, 王同仁2, 张祥林3.枣疯病植原体TaqMan探针实时荧光定量PCR检测方法的建立[J].植物保护,2014,40(5):111-116. |
| 枣疯病植原体TaqMan探针实时荧光定量PCR检测方法的建立 |
| Establishment of real-time fluorescent quantitative PCR method with TaqMan probe for detection of jujube witches’ broom phytoplasma |
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| DOI： |
| 中文关键词: 枣疯病植原体 TaqMan探针 实时荧光定量PCR 检测 |
| 英文关键词:jujube witches’ broom phytoplasma TaqMan probe real-time fluorescent quantitative PCR detection |
| 基金项目:新疆维吾尔自治区高校科研计划项目(XJEDU2010S15); 新疆维吾尔自治区自然科学基金(2010211A17); 质检公益性行业科研专项(201310091) |
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| 中文摘要: |
| 根据枣疯病植原体16S rDNA基因保守区域设计、合成特异性引物和TaqMan探针, 以构建的重组质粒作为阳性标准品, 建立并优化了对枣疯病植原体的TaqMan实时荧光定量PCR检测方法。对优化后的方法进行灵敏度、特异性及稳定性评价, 制作了标准曲线。结果显示, 制作的标准曲线有极好的线性关系, 相关系数( r 2 )达到0.998, 建立的实时荧光定量PCR检测方法能够特异性地检测枣疯病植原体, 能检测到60拷贝的质粒DNA。本研究建立的实时荧光定量PCR检测方法灵敏度、特异性、重复性好, 不仅能够实现对枣疯病植原体的快速检测, 而且为实现从病原定量水平上对枣疯病病情分级奠定了基础。 |
| 英文摘要: |
| A set of primers and TaqMan probe specific for Jujube witches’ broom phytoplasma were designed according to the conserved region of 16S rDNA gene, and the recombinant plasmid was constructed as a standard control. A TaqMan real-time fluorescent PCR assay for quantitative detection of the pathogen was established and optimized. The evaluation assay indicated that a good linear correlation was demonstrated in the standard curve for the real-time fluorescent PCR assay, with the correlation coefficient ( r2 ) of 0.998. The method was specific to jujube witches’ broom phytoplasma, and it can detect 60 copies/μL plasmid DNA. It not only provides a sensitive, specific and reproducible method for detection of jujube witches’ broom phytoplasma, but also lays the foundation for the grading system of the pathogen at quantitative level. |
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