Rapid detection of fig mosaic virus using reverse transcription loop-mediated isothermal amplification
Rapid detection of fig mosaic virus using reverse transcription loop-mediated isothermal amplification
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DOI：
10.1007/s10327-015-0603-1
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发表时间：
2015-09-01
影响因子：
1.2
通讯作者：
Namba, Shigetou
中科院分区：
文献类型：
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作者：
Ishikawa, Kazuya;Maejima, Kensaku;Namba, Shigetou
无花果花叶病毒（FMV）是一种负链RNA病毒，是引起无花果花叶病毒病的病原，在世界上几乎所有的无花果种植国均有发生，严重影响了世界范围内的无花果生产。使用FMV特异性引物的逆转录-聚合酶链反应（RT-PCR）通常用于检测FMV，但需要更快和更容易的FMV检测方法，因为RT-PCR需要多个步骤和特殊设备。本研究建立了一种检测FMV的逆转录环介导等温扩增（RT-LAMP）方法。我们根据FMV分离株之间的序列保守性设计了LAMP引物。RT-LAMP反应使用的引物组靶向RNA 3从FMV感染的无花果叶提取的总RNA在15分钟内导致快速检测。此外，我们建立了一个快速，简单的方法，直接从无花果叶使用木制牙签采样。当使用直接取样方法时，RT-LAMP反应特异性和反应性几乎与使用分离的总RNA获得的那些相当。此外，使用FMV RT-LAMP测定可检测到地理上和遗传学上不同的FMV分离株。本试验为FMV的检测提供了一种实用的方法。
Fig mosaic virus (FMV), a negative-strand RNA virus, is a causal agent of fig mosaic disease, which occurs in almost all countries where figs are grown and severely affects worldwide fig production. Reverse transcription-polymerase chain reaction (RT-PCR) using FMV-specific primers has typically been used to detect FMV, but faster and easier detection methods for FMV are required because RT-PCR requires multiple steps and special equipment. In this study, we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to detect FMV. We designed LAMP primer sets based on sequence conservation among FMV isolates. The RT-LAMP reaction using a primer set targeting RNA3 in total RNA extracted from FMV-infected fig leaves resulted in rapid detection within 15 min. In addition, we established a fast and simple method of direct sampling from fig leaves using a wooden toothpick. The RT-LAMP reaction specificity and reactivity when using the direct sampling method were almost comparable to those obtained using isolated total RNA. Moreover, geographically and phylogenetically distinct FMV isolates were detectable using the FMV RT-LAMP assay. The assay presented here provides a practical method to detect FMV.