建立环介导等温扩增检测方法快速检测.
Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of .
机构信息
Department of Plant Pathology, University of Georgia, Tifton, GA 31793, USA.
U. S. Vegetable Laboratory, USDA-ARS, Charleston, SC 29414, USA.
出版信息
Int J Mol Sci. 2020 Mar 4;21(5):1756. doi: 10.3390/ijms21051756.
A loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of (CuLCrV), one of the most important begomoviruses that infects cucurbits worldwide. A set of six specific primers targeting a total 240 nt sequence regions in the DNA A of CuLCrV were designed and synthesized for detection of CuLCrV from infected leaf tissues using real-time LAMP amplification with the Genie III system, which was further confirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. The optimum reaction temperature and time were determined, and no cross-reactivity was seen with other begomoviruses. The LAMP assay could amplify CuLCrV from a mixed virus assay. The sensitivity assay demonstrated that the LAMP reaction was more sensitive than conventional PCR, but less sensitive than qPCR. However, it was simpler and faster than the other assays evaluated. The LAMP assay also amplified CuLCrV-infected symptomatic and asymptomatic samples more efficiently than PCR. Successful LAMP amplification was observed in mixed virus-infected field samples. This simple, rapid, and sensitive method has the capacity to detect CuLCrV in samples collected in the field and is therefore suitable for early detection of the disease to reduce the risk of epidemics.
建立了一种环介导等温扩增(LAMP)检测方法,用于简单、快速、高效地检测(CuLCrV),这是一种在全球范围内感染葫芦科植物的最重要的番茄黄曲叶病毒之一。设计并合成了一套针对 CuLCrV DNA A 区全长 240nt 序列的 6 条特异性引物,用于通过 Genie III 系统实时 LAMP 扩增检测 CuLCrV,进一步通过凝胶电泳和 SYBR™ Green I DNA 染色进行视觉观察进行了确认。确定了最佳反应温度和时间,并且与其他番茄黄曲叶病毒没有交叉反应。LAMP 检测可以从混合病毒检测中扩增出 CuLCrV。敏感性检测表明,LAMP 反应比常规 PCR 更敏感,但比 qPCR 不敏感。然而,它比评估的其他检测方法更简单、更快。LAMP 检测也比 PCR 更有效地扩增 CuLCrV 感染的有症状和无症状样本。在混合病毒感染的田间样本中成功观察到 LAMP 扩增。这种简单、快速、灵敏的方法能够在田间采集的样本中检测到 CuLCrV,因此适合早期检测疾病,降低流行风险。