| 杨雷亮,管维,孙丽霞,等.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,(6-7):- |
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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 the Fluorescent LAMP Detection System for the pathogen of Moko disease |
| 投稿时间：2020-04-16 修订日期：2020-05-17 |
| DOI： |
| 中文关键词: 香蕉细菌性枯萎病 实时荧光LAMP 定性检测 |
| 英文关键词: Moko disease Real-time fluorescence LAMP qualitative detection |
| 基金项目:拱北海关科技项目：香蕉细菌性枯萎病菌荧光LAMP检测技术研究(2018GDK27),中山市社会公益科技研究项目：基于16S rRNA基因二代高通量测序法分析中山市PM2.5细菌群落特征(2017B1161) |
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| 中文摘要: |
| [目的] 基于香蕉细菌性枯萎病病原（植物青枯菌）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小时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，采用了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了试验。[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、特异性及该方法的可重复性强，灵敏度高出普通PCR10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: |
| [Objcetive]The purpose of the study is To establish a real-time fluorescence qualitative detection method based on the LAMP technology and the specific conserved primers of the pathogen of Moko disease(Ralstonia solanacearum ,race2) . As the low specificity, low sensitivity, and long time-consuming for qualitative detection of conventional PCR, an accurate and efficient qualitative detecting within an hour can effectively replace it and provide technical support for the work on the protecting and controlling detection and the quarantine of the disease. [Mehtod] Determine the sequence of target gene and use the Primer software to design the internal and external primers. The LAMP reaction system consists of two pairs of primers, DNA polymerase with the characteristic of strand displacement,template DNA, betaine, Mg2SO4, reaction buffer, fluorescent dye, etc.A lid-opening detection can be avoided after the reaction and the sensitivity, specificity and repeatability of this method for the qualitative detection of sample DNA can be validated effectively. [Result] A The designed primers can successfully amplify the target gene.The real-time fluorescent LAMP detection system has good sensitivity ,specificity and strong repeatability for the pathogen of Moko disease.The sensitivity of this method is 10 times more higher than the ordinary PCR,but not as much as the HRCA(Hyper-branched rolling cycle amplification). [Conclusion] Establishment of the Fluorescent LAMP Detection System for the pathogen of Moko disease shew high specificity, high sensitivity, low possibility of pollution and short time-consuming for qualitative detection within an hour. |
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