Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil.
Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil.
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DOI：
10.1371/journal.pone.0082841
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Peng J
中科院分区：
文献类型：
--
作者：
Zhang X;Zhang H;Pu J;Qi Y;Yu Q;Xie Y;Peng J
香蕉枯萎香蕉枯萎病（Fusarium wilt，Panama disease）是香蕉（Musa spp.）Foc热带小种4（TR 4）目前被认为是全球香蕉生产中的主要问题。目前尚不清楚芭蕉属植物对Foc有有效的抗性，而且一旦香蕉植物受到感染，也没有有效的措施来控制Foc。早期准确检测Foc TR 4对保护香蕉产业和指导香蕉种植至关重要。建立了一种实时荧光环介导等温扩增法（real-time fluorescence loop-mediated isothermal amplification assay，RealPCR），用于土壤中Foc TR 4的快速定量检测。当与提取的土壤DNA或103个孢子/g人工感染土壤混合时，Realacidin检测方法的检测限约为0.4 pg/µl质粒DNA，与其他相关病原体无交叉反应。通过测试人工和天然侵染的样品来确认用于定量TR 4基因组DNA的Realtase测定。Foc TR 4对自然感染样品中土传病原菌DNA的定量结果与经典的实时荧光定量PCR相比差异不显著（P>0.05）。此外，Realtase测定是通过在扩增之前将SYBR绿色I荧光染料添加到盖的内部而用改进的闭管视觉检测系统视觉化的，这避免了染料对DNA扩增的抑制作用，并且使得该测定在现场更方便，因此可以成为简单的，一种快速有效的技术，有可能作为一种替代工具，用于现场检测和监测Foc TR 4，这将是一个例行的DNA为基础的测试服务，为土壤传播的病原体在中国南方。
Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 103 spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China.
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影响因子：
3.7
作者：
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通讯作者：
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