喜树丛枝植原体的分子鉴定及TaqMan探针实时荧光定量PCR检测方法的建立
Identification of Camptotheca acuminate witches′-broom phytoplasma and TaqMan-based fluorescence qPCR method for detection
查看参考文献27篇
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文摘
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为确定在云南文山地区喜树上发生的疑似丛枝病的病原种类及快速检测喜树丛枝病,本研究利用植原体16S rDNA基因通用引物P1/P7和R16F2n/R16R2对感病喜树总DNA进行常规PCR和巢式PCR扩增、克隆和测序,通过系统进化分析,明确了喜树丛枝植原体属于16SrXXXII组。然后根据喜树丛枝病植原体16S rDNA基因保守区域设计并合成特异性引物和TaqMan探针,制备了喜树丛枝病植原体标准质粒,确定了最优引物浓度和最佳探针浓度,制作的标准曲线有极好的线性关系,决定系数(R~2)达到0.999,建立的实时荧光定量PCR检测方法能够特异性地检测喜树丛枝植原体。本研究首次明确了喜树丛枝植原体的分类地位,优化和建立了喜树丛枝植原体TaqMan探针qPCR检测方法,为快速检测喜树丛枝病植原体提供参考。 |
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其他语种文摘
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Camptotheca acuminata witches′-broom-like disease was founded in Wenshan of Yunnan Province. To determine the pathogen and develop rapidly detection approach, near-full-length phytoplasmal 16S rDNA gene sequences were amplified via conventional PCR and nested-PCR using universal primer pairs P1/P7, R16F2n/R16R2 from DNAs extracted from the symptomatic shoots and leaves of C. acuminata plants. The amplicons were cloned and sequenced. Phylogenetic analysis indicates that the pathogen is a member of phytoplasma group 16SrXXXII. Then, a set of primers and TaqMan probe specific for the phytoplasma were designed according to the conserved region of 16S rDNA gene. The standard plasmid DNA of Camptotheca acuminata witches′broom phytoplasma was prepared, and the concentrations of primers and probe were optimized. The evaluation assay indicated that a good linear correlation was demonstrated in the standard curve with the correlation coefficient (r~2 ) of 0.999. The established real-time fluorescence quantitative PCR detection method could specifically detected the phytoplasma in witches′-broom-like disease in C. acuminata. To our best knowledge, it is the first report of a 16SrII group phytoplasma associated with witches′-broom disease in C. acuminata in Yunnan Province. And it is the first study to determine the pathogen of Camptotheca acuminata witches′-broom disease, and a TaqMan probe-based qPCR method was established for the pathogen detection of this disease. The results of this study can provide reference for rapid detection and identification of Camptotheca acuminata witches′broom phytoplasma. |
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来源
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植物病理学报
,2021,51(3):429-440 【核心库】
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DOI
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10.13926/j.cnki.apps.000707
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关键词
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喜树丛枝病
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植原体
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TaqMan探针
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实时荧光定量PCR
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地址
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1.
云南农业大学, 农业生物多样性与病虫害控制教育部重点实验室, 昆明, 650201
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文山州农业科学院, 文山, 663000
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玉溪师范学院资源环境学院, 玉溪, 653100
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语种
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中文 |
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文献类型
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研究性论文 |
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ISSN
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0412-0914 |
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学科
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植物保护 |
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基金
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国家自然科学基金项目
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云南省教育厅科研基金
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文献收藏号
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CSCD:7015170
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