One-step reverse transcription loop-mediated isothermal amplification for the rapid detection of cucumber green mottle mosaic virus
One-step reverse transcription loop-mediated isothermal amplification for the rapid detection of cucumber green mottle mosaic virus
复制标题
一步逆转录环介导等温扩增快速检测黄瓜绿斑驳花叶病毒
DOI：
10.1016/j.jviromet.2013.07.059
复制
发表时间：
2013-11-01
影响因子：
3.1
通讯作者：
Tao, Xiao-rong
中科院分区：
文献类型：
--
作者：
Li, Jin-yu;Wei, Qi-wei;Tao, Xiao-rong
黄瓜绿斑驳花叶病毒(CGMMV)已对世界各地的葫芦科作物造成严重危害。该病毒被认为是许多国家最严重的葫芦科检疫原因之一。本研究建立了一种高效、实用的一步反转录环介导等温扩增技术(RT-LAMP)，用于检测烟草花叶病毒。该RT-LAMP方法可以方便地检测到从西瓜植株或种子中提取的总RNA或粗RNA。RT-LAMP检测在63℃恒温条件下进行1h，琼脂糖凝胶电泳法可检测到CGMMV扩增产物呈梯状条带，或在紫外光照射下加入荧光染料可在试管内观察到CGMMV扩增产物。RT-LAMP扩增是针对CGMMV的，与其他病毒未见交叉反应。RT-LAMP检测的灵敏度是逆转录-聚合酶链式反应(RT-PCR)的100倍。这是首次应用RT-LAMP方法检测CGMMV。本研究建立的灵敏、特异、快速的RT-LAMP方法可广泛应用于实验室、田间和检疫监测中。(C)2013爱思唯尔B.V.保留所有权利。
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 degrees C) conditions within 1 h. 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. (C) 2013 Elsevier B.V. All rights reserved.