Real-time detection of cucurbit chlorotic yellows virus by RT-LAMP

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods Pub Date : 2025-09-09 DOI:10.1016/j.jviromet.2025.115262

Ángela Martínez-Fernández , Josemaría Delgado-Martín , Esmeralda Sanahuja-Edo , Ana Alfaro-Fernández , José Manuel Estévez , Isabel Font-San-Ambrosio , Luis Galipienso , Luis Rubio

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引用次数: 0

Abstract

Cucurbit chlorotic yellows virus (CCYV) of the genus Crinivirus and family Closteroviridae, is an emerging infectious agent transmitted by whiteflies that mainly infects cucumber, melon and watermelon plants. Here, we developed a procedure based on reverse transcription (RT) followed by loop-mediated isothermal amplification (LAMP) to detect CCYV in real-time by using a fluorescent dye and incubating at 65 °C. This method detected CCYV in RNA extracts at about 10 min and in nylon membrane-filtered crude extracts between 55 and 95 min. The detection was sensitive, ten times higher than RT followed by polymerase chain reaction (PCR) and specifically detecting different isolates of CCYV without cross-reaction with other viruses of the same genus. This procedure enables the simultaneous analyses of multiple samples, allowing for rapid, sensitive and specific detection of CCYV. By using membrane-filtered crude extracts, RNA purification is unnecessary, so the whole process can be performed in the field with a portable heater and fluorometer, facilitating a rapid response for disease control.

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RT-LAMP实时检测瓜绿黄病毒。

葫芦绿黄病毒（Cucurbit chlorotic yellow virus， CCYV）是一种由白蝇传播的新型传染病，主要感染黄瓜、甜瓜和西瓜等植物。在这里，我们开发了一种基于逆转录（RT）和环介导等温扩增（LAMP）的程序，通过荧光染料和65°C孵育实时检测CCYV。该方法在RNA提取物中检测CCYV约10min，在尼龙膜过滤的粗提取物中检测55 ~ 95min。该方法灵敏度高，比RT法和聚合酶链反应（PCR）法高10倍，能特异性检测不同分离株，且不与同一属其他病毒发生交叉反应。该程序可以同时分析多个样品，允许快速，敏感和特异性检测CCYV。采用膜过滤粗提物，无需RNA纯化，整个过程可在现场使用便携式加热器和荧光仪进行，便于快速响应疾病控制。

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

Journal of virological methods 医学-病毒学

CiteScore

5.80

自引率

0.00%

发文量

209

审稿时长

41 days

期刊介绍： The Journal of Virological Methods focuses on original, high quality research papers that describe novel and comprehensively tested methods which enhance human, animal, plant, bacterial or environmental virology and prions research and discovery. The methods may include, but not limited to, the study of: Viral components and morphology- Virus isolation, propagation and development of viral vectors- Viral pathogenesis, oncogenesis, vaccines and antivirals- Virus replication, host-pathogen interactions and responses- Virus transmission, prevention, control and treatment- Viral metagenomics and virome- Virus ecology, adaption and evolution- Applied virology such as nanotechnology- Viral diagnosis with novelty and comprehensive evaluation. We seek articles, systematic reviews, meta-analyses and laboratory protocols that include comprehensive technical details with statistical confirmations that provide validations against current best practice, international standards or quality assurance programs and which advance knowledge in virology leading to improved medical, veterinary or agricultural practices and management.