| 杨雷亮 1,2，管 维 1，孙丽霞 2，吴颖儿 1，单振菊 1，王章根 1，陈定虎 1.香蕉细菌性枯萎病菌荧光 LAMP 检测体系的建立[J].广东农业科学,2020,47(5):73- |
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香蕉细菌性枯萎病菌荧光 LAMP 检测体系的建立 |
| Establishment of Fluorescence LAMP Detection System for Pathogen of Moko Disease |
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| DOI：10.16768/j.issn.1004-874X.2020.05.009 |
| 中文关键词: 香蕉枯萎病 细菌 青枯菌 检测 环介导等温扩增（LAMP） 实时荧光 |
| 英文关键词: banana bacterial wilt disease bacteria Ralstonia solanacearum detection loop-mediated isothermal amplification（LAMP） real-time fluorescence |
| 基金项目:66 |
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| 中文摘要: |
| 【目的】基于香蕉细菌性枯萎病病原（Ralstonia solanacearum）2 号生理小种的特异保守引物和LAMP 技术建立一种实时荧光定性检测方法，解决常规 PCR 定性检测特异性低、灵敏度低、耗时长等问题，实现在 1 h 内能进行准确高效的定性检测，为加强该病害防控检测和检疫工作提供技术支持。【方法】确定被测基因靶序列，利用 Primer 软件设计两对内外引物。由两对引物、具有链置换特性的 DNA 聚合酶、模板 DNA、甜菜碱、Mg2SO4、反应缓冲液、荧光染料等组成 LAMP 反应体系，采用不必在反应完开管盖检测的实时荧光 LAMP 方法，针对该方法对于样品 DNA 定性检测的灵敏度、特异性及可重复性进行试验。【结果】设计的引物能顺利扩增目标基因，实时荧光 LAMP 检测体系对于香蕉细菌性枯萎病病原生理 2 号小种有良好的灵敏度、特异性，该方法可重复性强，灵敏度比普通 PCR 高 10 倍以上。【结论】香蕉细菌性枯萎病菌荧光 LAMP 检测体系检测特异性强、灵敏度高、耗时短、不易污染，效果较好。 |
| 英文摘要: |
| 【Objective】The purpose of the study is to establish a real-time fluorescence qualitative detection method based on the LAMP technology and the specific conservative primers of the pathogen of Moko disease（Ralstonia solanacearum, race 2）. To solve the problems such as low specificity, low sensitivity and long time-consuming of qualitative detection of conventional PCR, an accurate and efficient qualitative detection within an hour can be achieved, which will provide technical support for the detection and quarantine of prevention and control of the disease.【Method】The sequence of gene target was determined and the Primer software was used to design two pairs of internal and external primers. The LAMP reaction system consisted of two pairs of primers, DNA polymerase with characteristics of strand displacement, template DNA, betaine, Mg 2SO4, reaction buffer, fluorescent dye etc. By using real-time fluorescence LAMP method, a lid-opening detection could be avoided after reaction and the sensitivity, specificity and repeatability of this method for the qualitative detection of sample DNA could be validated effectively.【Result】The designed primers could successfully amplify the target gene. The real-time fluorescence LAMP detection system had good sensitivity with specificity and strong repeatability for the pathogen of Moko disease, which was 10 times higher than that of ordinary PCR.【Conclusion】 The fluorescence LAMP detection system shows high specificity, high sensitivity, short time-consuming and low possibility of pollution for the detection of the pathogen of Moko disease. |
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