Development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Hemileia vastatrix in coffee plantations

IF 2.5 3区农林科学 Q2 Agricultural and Biological Sciences Tropical Plant Pathology Pub Date : 2024-03-15 DOI:10.1007/s40858-023-00627-z

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Abstract

Coffee leaf rust (CLR) caused by Hemileia vastatrix is a devastating worldwide disease. Early monitoring is crucial for controlling CLR quickly and efficiently. However, accurately identifying CLR in its early stages via the naked eye is challenging. Moreover, detecting H. vastatrix using PCR-based methods is time-consuming, labour-intensive, and occasionally exhibits low sensitivity. Loop-Mediated Isothermal Amplification (LAMP) technology is known for its speed, specificity, and sensitivity to identifying many pathogens accurately. Therefore, in this study, we conducted a comparative analysis of ITS sequences from H. vastatrix and other H. vastatrix and Uredinales strains available in the National Center for Biotechnology Information (NCBI) database using the BLASTn tool. Based on this analysis, we designed specific primers that target the unique region and its flanking regions within the ITS sequences of H. vastatrix. Using SYBR Green I dye, we established a LAMP technique for rapid and sensitive detection of H. vastatrix. Moreover, we optimised the LAMP protocol to enhance sensitivity and specificity for H. vastatrix detection. Under the optimised conditions, the established LAMP protocol detected as little as 1pg/μL of H. vastatrix DNA within 60min at 63°C. This sensitivity is approximately 100 times higher than that achieved using conventional PCR. Our method proved effective in detecting H. vastatrix at the early stages of CLR symptom development on the coffee leaves in field conditions.

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开发环介导等温扩增测定法，用于快速、灵敏地检测咖啡种植园中的 Hemileia vastatrix

摘要由 Hemileia vastatrix 引起的咖啡叶锈病（CLR）是一种毁灭性的世界性病害。早期监测对于快速有效地控制 CLR 至关重要。然而，通过肉眼准确识别早期阶段的 CLR 具有挑战性。此外，使用基于 PCR 的方法检测 H. vastatrix 既费时又费力，有时灵敏度还很低。环路介导等温扩增（LAMP）技术以其快速、特异性和灵敏度高而著称，可准确识别多种病原体。因此，在本研究中，我们使用 BLASTn 工具对 H. vastatrix 和美国国家生物技术信息中心（NCBI）数据库中的其他 H. vastatrix 和 Uredinales 菌株的 ITS 序列进行了比较分析。在此分析基础上，我们设计了针对 H. vastatrix ITS 序列中独特区域及其侧翼区域的特异引物。利用 SYBR Green I 染料，我们建立了一种快速灵敏检测 H. vastatrix 的 LAMP 技术。此外，我们还对 LAMP 方案进行了优化，以提高检测 H. vastatrix 的灵敏度和特异性。在优化的条件下，所建立的 LAMP 方案可在 63°C 下 60 分钟内检测到低至 1pg/μL 的 H. vastatrix DNA。这一灵敏度比传统 PCR 方法高出约 100 倍。事实证明，在田间条件下，我们的方法能在咖啡叶片上出现 CLR 症状的早期阶段有效地检测出 H. vastatrix。

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来源期刊

Tropical Plant Pathology PLANT SCIENCES-

CiteScore

4.50

自引率

4.00%

发文量

审稿时长

6-12 weeks

期刊介绍： Tropical Plant Pathology is an international journal devoted to publishing a wide range of research on fundamental and applied aspects of plant diseases of concern to agricultural, forest and ornamental crops from tropical and subtropical environments.  Submissions must report original research that provides new insights into the etiology and epidemiology of plant disease as well as population biology of plant pathogens, host-pathogen interactions, physiological and molecular plant pathology, and strategies to promote crop protection. The journal considers for publication: original articles, short communications, reviews and letters to the editor. For more details please check the submission guidelines. Founded in 1976, the journal is the official publication of the Brazilian Phytopathology Society.