Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, China.
J Virol Methods. 2013 Nov;193(2):583-8. doi: 10.1016/j.jviromet.2013.07.059. Epub 2013 Aug 6.
Cucumber green mottle mosaic virus (CGMMV) has caused serious damage to Cucurbitaceae crops worldwide. The virus is considered one of the most serious Cucurbitaceae quarantine causes in many countries. In this study, a highly efficient and practical one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of CGMMV. The total RNA or crude RNA extracted from watermelon plants or seeds could be detected easily by this RT-LAMP assay. The RT-LAMP assay was conducted in isothermal (63°C) conditions within 1h. The amplified products of CGMMV could be detected as ladder-like bands using agarose gel electrophoresis or visualized in-tube under UV light with the addition of a fluorescent dye. The RT-LAMP amplification was specific to CGMMV, as no cross-reaction was observed with other viruses. The RT-LAMP assay was 100-fold more sensitive than that of reverse-transcription polymerase chain reaction (RT-PCR). This is the first report of the application of the RT-LAMP assay to detect CGMMV. The sensitive, specific and rapid RT-LAMP assay developed in this study can be applied widely in laboratories, the field and quarantine surveillance of CGMMV.
黄瓜绿斑驳花叶病毒（CGMMV）已在全球范围内对葫芦科作物造成严重危害。该病毒被认为是许多国家葫芦科检疫的最严重原因之一。在这项研究中，开发了一种高效实用的一步法逆转录环介导等温扩增（RT-LAMP）技术，用于检测 CGMMV。该 RT-LAMP 检测方法可以轻松检测从西瓜植株或种子中提取的总 RNA 或粗 RNA。RT-LAMP 反应在等温（63°C）条件下 1 小时内完成。通过琼脂糖凝胶电泳或添加荧光染料后在管内观察到紫外线，可以检测到 CGMMV 的扩增产物呈梯状条带。RT-LAMP 扩增对 CGMMV 具有特异性，因为与其他病毒没有交叉反应。RT-LAMP 检测比逆转录聚合酶链反应（RT-PCR）灵敏 100 倍。这是首次应用 RT-LAMP 检测 CGMMV。本研究中开发的灵敏、特异和快速 RT-LAMP 检测方法可广泛应用于 CGMMV 的实验室、现场和检疫监测。
