Rapid real-time quantitative colorimetric LAMP methodology for field detection of Verticillium dahliae in crude olive-plant samples

IF 4.7 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Plant Methods Pub Date : 2024-09-09 DOI:10.1186/s13007-024-01251-x

Maria Megariti, Alexandra Panagou, Georgios Patsis, George Papadakis, Alexandros K. Pantazis, Epaminondas J. Paplomatas, Aliki K. Tzima, Emmanouil A. Markakis, Electra Gizeli

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引用次数: 0

Abstract

Verticilium dahliae is the most important wilt pathogen of olive trees with a broad host range causing devastating diseases currently without any effective chemical control. Traditional detection methodologies are based on symptoms-observation or lab-detection using time consuming culturing or molecular techniques. Therefore, there is an increasing need for portable tools that can detect rapidly V. dahliae in the field. In this work, we report the development of a novel method for the rapid, reliable and on-site detection of V. dahliae using a newly designed isothermal LAMP assay and crude extracts of olive wood. For the detection of the fungus, LAMP primers were designed targeting the internal transcribed spacer (ITS) region of the rRNA gene. The above assay was combined with a purpose-built prototype portable device which allowed real time quantitative colorimetric detection of V. dahliae in 35 min. The limit of detection of our assay was found to be 0.8 fg/μl reaction and the specificity 100% as indicated by zero cross-reactivity to common pathogens found in olive trees. Moreover, detection of V. dahliae in purified DNA gave a sensitivity of 100% (Ct < 30) and 80% (Ct > 30) while the detection of the fungus in unpurified crude wood extracts showed a sensitivity of 80% when multisampling was implemented. The superiority of the LAMP methodology regarding robustness and sensitivity was demonstrated when only LAMP was able to detect V. dahliae in crude samples from naturally infected trees with very low infection levels, while nested PCR and SYBR qPCR failed to detect the pathogen in an unpurified form. This study describes the development of a new real time LAMP assay, targeting the ITS region of the rRNA gene of V. dahliae in olive trees combined with a 3D-printed portable device for field testing using a tablet. The assay is characterized by high sensitivity and specificity as well as ability to operate using directly crude samples such as woody tissue or petioles. The reported methodology is setting the basis for the development of an on-site detection methodology for V. dahliae in olive trees, but also for other plant pathogens.

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现场检测橄榄植株粗样品中大丽轮枝菌的快速实时定量比色 LAMP 方法

大丽轮枝菌（Verticilium dahliae）是橄榄树最重要的枯萎病病原体，寄主范围很广，会造成毁灭性病害，目前还没有有效的化学防治方法。传统的检测方法基于症状观察，或使用耗时的培养或分子技术进行实验室检测。因此，越来越需要能在田间快速检测大丽花病毒的便携式工具。在这项工作中，我们报告了利用新设计的等温 LAMP 检测法和橄榄木的粗提取物，开发出一种快速、可靠和现场检测大丽花病毒的新方法。为检测该真菌，设计了针对 rRNA 基因内部转录间隔区（ITS）的 LAMP 引物。上述检测方法与专门设计的便携式原型设备相结合，可在 35 分钟内对大丽花病毒进行实时定量比色检测。我们发现该检测方法的检测限为 0.8 fg/μl，特异性为 100%，因为它与橄榄树中常见病原体的交叉反应为零。此外，在纯化的 DNA 中检测大丽花病毒的灵敏度为 100%（Ct 30），而在未纯化的粗木材提取物中检测该真菌的灵敏度为 80%。LAMP 方法在稳健性和灵敏度方面的优越性得到了证明：在感染水平很低的自然感染树木的粗样品中，只有 LAMP 能够检测到大丽花病毒，而嵌套 PCR 和 SYBR qPCR 则无法检测到未纯化形式的病原体。本研究介绍了一种新型实时 LAMP 检测方法的开发情况，该方法以橄榄树中大丽花病毒 rRNA 基因的 ITS 区域为检测目标，并结合了一种 3D 打印便携式设备，可使用平板电脑进行现场检测。该检测方法的特点是灵敏度高、特异性强，能够直接使用木质组织或叶柄等粗样品进行操作。所报告的方法为开发现场检测橄榄树中大丽花病毒以及其他植物病原体的方法奠定了基础。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Plant Methods 生物-植物科学

CiteScore

9.20

自引率

3.90%

发文量

121

审稿时长

2 months

期刊介绍： Plant Methods is an open access, peer-reviewed, online journal for the plant research community that encompasses all aspects of technological innovation in the plant sciences. There is no doubt that we have entered an exciting new era in plant biology. The completion of the Arabidopsis genome sequence, and the rapid progress being made in other plant genomics projects are providing unparalleled opportunities for progress in all areas of plant science. Nevertheless, enormous challenges lie ahead if we are to understand the function of every gene in the genome, and how the individual parts work together to make the whole organism. Achieving these goals will require an unprecedented collaborative effort, combining high-throughput, system-wide technologies with more focused approaches that integrate traditional disciplines such as cell biology, biochemistry and molecular genetics. Technological innovation is probably the most important catalyst for progress in any scientific discipline. Plant Methods’ goal is to stimulate the development and adoption of new and improved techniques and research tools and, where appropriate, to promote consistency of methodologies for better integration of data from different laboratories.