State University of Ponta Grossa, Department of Plant Science and Plant Protection, Uvaranas, 84030-900, Ponta Grossa, PR, Brazil.
State University of Maringá, Department of Agronomy, Jardim Universitário, 87020-900, Maringá, PR, Brazil.
Plant Dis. 2020 May;104(5):1514-1526. doi: 10.1094/PDIS-07-19-1455-RE. Epub 2020 Feb 26.
is one of the most devastating and cosmopolitan plant pathogens. Rapid detection of can provide growers an advantage in knowing what control measures should be taken to minimize crop damage and financial losses caused by it. Loop-mediated isothermal amplification (LAMP) is a fast, sensitive, and specific nucleic acid amplification method that does not require a thermal cycler. This study aimed to develop a LAMP-based assay for the specific detection of (Ss-LAMP). A real-time quantitative LAMP reaction (Ss-qLAMP) and a calcein ion indicator-based LAMP reaction (Ss-cLAMP) were designed, optimized, and tested on fungi, plant, and soil samples. The Ss-LAMP reactions were very specific and sensitive. Applying the artificially inoculated soil samples with DNA purified by five protocols in the Ss-qLAMP reaction, it was possible to detect and quantify the pathogen DNA, regardless of the extraction protocol. Naturally infected soybean tissues had the pathogen detected by Ss-cLAMP directly in the reaction tube with no DNA extraction requirement. The assays should be applicable for many types of samples, such as soil, spore traps, and plant tissues from several crops, with no requirement for DNA extraction. The Ss-LAMP reactions took less than 1 h to complete, and they can be made directly in the field with real-time quantitative results (Ss-qLAMP) or qualitative naked-eye visual results (Ss-cLAMP). Results were obtained with 10 pg of DNA or 10 ng of crude mycelium, suggesting a detection limit close to a single DNA copy. Ss-LAMP reactions will allow rapid and accurate diagnosis of and assist in pathogen management and control.
是一种极具破坏力且分布广泛的植物病原体。快速检测 可以让种植者提前了解应采取哪些控制措施，从而将其对作物造成的损害和经济损失降到最低。环介导等温扩增（LAMP）是一种快速、敏感、特异的核酸扩增方法，不需要热循环仪。本研究旨在开发一种基于 LAMP 的检测方法，用于特异性检测 （Ss-LAMP）。设计并优化了实时定量 LAMP 反应（Ss-qLAMP）和钙黄绿素离子指示剂 LAMP 反应（Ss-cLAMP），并在真菌、植物和土壤样本中进行了测试。Ss-LAMP 反应非常特异和敏感。在 Ss-qLAMP 反应中，应用五种不同方案提取的 DNA 对人工接种土壤样本进行分析，均能检测和定量分析病原体 DNA，而不受提取方案的影响。无需提取 DNA，即可直接在反应管中用 Ss-cLAMP 检测到自然感染大豆组织中的病原体。该检测方法可应用于多种类型的样本，如土壤、孢子陷阱和几种作物的植物组织，无需提取 DNA。Ss-LAMP 反应在 1 小时内完成，并且可以在现场直接进行，实时定量结果（Ss-qLAMP）或定性肉眼观察结果（Ss-cLAMP）。该检测方法的检测限接近单个 DNA 拷贝，只需 10 pg 的 DNA 或 10 ng 的粗菌丝即可获得结果。Ss-LAMP 反应将能够快速准确地诊断 ，并有助于对病原体进行管理和控制。
