| 摘要: |
| 【目的】建立葡萄霜霉病菌（Plasmopara viticola）重组酶聚合酶扩增 侧流层析试纸条（recombinase polymerase amplification-lateral flow dipstick，LFD-RPA）检测体系，为快速准确诊断和有效防治葡萄霜霉病提供参考。【方法】依据葡萄霜霉病菌内转录间隔区（ITS）序列设计了PF1/PR1、PF2/PR2和PF3/PR3共3对引物及探针RPA-P，对葡萄霜霉病菌DNA的ITS序列进行PCR扩增，建立了葡萄霜霉病菌LFD-RPA检测体系，并对其反应条件进行优化，对其特异性和灵敏性进行检测。【结果】建立了葡萄霜霉病菌LFD-RPA检测体系，引物PF1/PR1和探针RPA-P对靶标片段具有良好的特异性，检测条件为36 ℃扩增30 min，对葡萄霜霉病菌具有特异性；最低检测下限为10 pg/μL，灵敏性略低于常规PCR(1 pg/μL)和荧光定量PCR（100 fg/μL）；对田间发病叶片的检测结果与常规PCR一致，准确性高。【结论】建立的葡萄霜霉病菌重组酶聚合酶扩增 侧流层析试纸条（LFD-RPA）检测体系，可以对目标物质进行快速、可视化检测，具有操作简单、特异性强、灵敏度高的特点，适宜推广使用。 |
| 关键词: 葡萄霜霉病 葡萄生单轴霉 重组酶聚合酶扩增-侧流层析试纸条检测 病原检测 |
| DOI：10.13207/j.cnki.jnwafu.2025.04.009 |
| 分类号: |
| 基金项目:宁夏回族自治区重大科技成果转化项目“酿酒葡萄病虫害信息化监测预警技术应用”（2023CJE09038） |
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| Establishment of the LFD-RPA detection system for Plasmopara viticola |
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HUANG Qiang, ZHANG Qiangqiang, GU Peiwen
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School of Agriculture,Ningxia University,Yinchuan,Ningxia 750021,China
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| Abstract: |
| 【Objective】The detection system of recombinase polymerase amplification combined with a lateral flow dipstick (LFD-RPA) assay for Plasmopara viticola was established to provide reference for rapid and accurate diagnosis and effective prevention and treatment of grape downy mildew.【Method】Based on the internal transcribed spacer (ITS) sequence of P.viticola,three pairs of primers (PF1/PR1,PF2/PR2 and PF3/PR3) and a probe (RPA-P) were designed, and the ITS sequence of P.viticola’s DNA was amplified by PCR,and the detection system of recombinase polymerase amplification-lateral flow dipstick (LFD-RPA) assay for P.viticola was established.The reaction conditions were optimized,and the specificity and sensitivity were detected.【Result】The LFD-RPA detection system of P. viticola was established.The primers PF1/PR1 and the probe RPA-P had good specificity to the target fragment.The detection condition was 36 °C amplification for 30 min,which was specific to P. viticola.The minimum detection limit was 10 pg/μL,and the sensitivity was slightly lower than that of conventional PCR (1 pg/μL) and Real-time PCR (100 fg/μL).The detection results of diseased leaves in the field were consistent with those of conventional PCR,which indicated the high accuracy of the detection system.【Conclusion】The established LFD-RPA detection system for P. viticola can be used for rapid and visual detection of target substances.With the characteristics of simple operation,strong specificity and high sensitivity,the system can be popularized and applied for further use. |
| Key words: grape downy mildew Plasmopara viticola recombinase polymerase amplification-lateral flow dipstick (LFD-RPA) detection detection of pathogens |
