向日葵黑茎病菌TaqMan-MGB探针实时荧光快速检测方法
Development of Taq Man-MGB fluorescent real-time PCR assay for the detection of Plenodomus lindquistii
查看参考文献38篇
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文摘
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向日葵黑茎病菌是我国进境检疫性有害生物名录中的一种检疫性真菌。根据向日葵黑茎病菌及其近似种的ITS序列差异,设计并合成特异性引物和探针,建立了向日葵黑茎病菌的实时荧光PCR检测方法。特异性试验结果表明,该检测方法能特异性检测向日葵黑茎病菌;灵敏度试验结果表明,最低检测限量为20 μL反应体系中总DNA含量0.1 pg;实时荧光PCR优化反应条件为引物终浓度0.6 μmol·L~(-1),探针终浓度0.3 μmol·L~(-1)。实际样品检测结果表明,该方法可用于疑似携带向日葵黑茎病菌样品的检测与初筛。此方法快速、灵敏,整个反应过程约1 h,检测过程完全闭管,无需PCR后续处理,为早期快速检测向日葵黑茎病菌提供了重要参考。 |
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其他语种文摘
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Plenodomus lindquistii is a quarantine fungus in the list of quarantine pests in China. A speciesspecific real-time polymerase chain reaction (PCR) assay was developed for the detection of P. lindquistii. A pair of specific primers and a TaqMan-MGB probe was designed and synthesized according to the difference of ITS sequence between P. lindquistii and related species. A novel real-time fluorescent PCR was established to detect P. lindquistii. The minimal detectable concentration of targeted DNA was 0.1 pg in 20 μL reaction mixture. Optimal primer concentration and probe concentration were 0.6 μmol·L~(-1) and 0.3 μmol·L~(-1),respectively. The method could be used for the detection and preliminary screening of the samples suspected of carrying P. lindquistii. The method was specific,rapid,sensitive and completed within a single tube,the whole reaction took about one hour,without post-PCR handling,which provided a valuable tool for early rapid detection and identification of P. lindquistii. |
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来源
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植物病理学报
,2021,51(6):975-986 【核心库】
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DOI
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10.13926/j.cnki.apps.000734
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关键词
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向日葵黑茎病菌
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实时荧光PCR
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TaqMan-MGB探针
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检测
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地址
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1.
宁波检验检疫科学技术研究院, 宁波, 315012
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宁波海关, 宁波, 315012
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中国科学院微生物研究所, 北京, 100101
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中国检验检疫科学研究院, 北京, 100176
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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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浙江省公益技术研究项目
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海关总署科研项目
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中国检科院科研项目
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文献收藏号
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CSCD:7159960
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