| 基于TaqMan MGB探针的黑白轮枝菌检测方法 |
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| 引用本文： | 郭立新,张祥林,陈先锋,段维军.基于TaqMan MGB探针的黑白轮枝菌检测方法[J].植物检疫,2017(1):32-36. |
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| 作者姓名： | 郭立新 张祥林 陈先锋 段维军 |
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| 作者单位： | 1. 宁波检验检疫科学技术研究院 浙江宁波315012;2. 新疆出入境检验检疫局 |
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| 基金项目： | 国家重点研发计划(2016YFF0203201)，浙江省博士后择优资助项目(BSH1402011)，宁波市科研项目(2015C110018&2014C50082) |
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| 摘 要： | 根据黑白轮枝菌(Verticillium albo-atrum)及其近似种β-微管蛋白基因(β-tubulin)序列差异,设计并合成1对引物和1条Taq Man-MGB探针,建立了黑白轮枝菌的实时荧光PCR检测方法。对供试黑白轮枝菌及其近似种实验表明,该方法特异性强,只有黑白轮枝菌可被检出。通过对反应体系的优化,确定了最佳反应条件:引物终浓度为1.0μmol/L,探针终浓度为0.7μmol/L。灵敏度试验结果显示,最低检测限量为总DNA含量10 pg(20μL反应体系)。此方法快速灵敏,为快速检测黑白轮枝菌提供了重要参考。
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| 关 键 词： | 黑白轮枝菌 实时荧光PCR TaqMan-MGB探针 检测 |
Development of TaqMan MGB fluorescent real-time PCR assay for the detection of Verticillium albo-atrum |
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| Guo Lixin,Zhang Xianglin,Chen Xianfeng,Duan Weijun.Development of TaqMan MGB fluorescent real-time PCR assay for the detection of Verticillium albo-atrum[J].Plant Quarantine,2017(1):32-36. |
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| Authors: | Guo Lixin Zhang Xianglin Chen Xianfeng Duan Weijun |
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| Abstract: | A pair of primers and a TaqMan-MGB probe based on the beta-tubulin sequence of V.albo-atrum and related isolates were designed and synthesized.A real-time fluorescent PCR was established to detect V.albo-atrum.The assay was validated with V.albo-atrum,it's phylogenetically most closely related species.Optimal primer concentration and probe concentration were 1.0 μmol/L and 0.7 μmol/L,respectively.The method detected as little as 10pg of total DNA in 20 μL reaction mixture.The method was rapid,sensitive and completed within a single tube,without post-PCR handling of the amplification products.The new method provides a valuable tool for early rapid detection and identification of V.albo-atrum. |
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| Keywords: | Verticillium albo-atrum real-time fluorescent PCR TaqMan-MGB probe detection |
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