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| 玉蜀黍黑粉菌环介导等温扩增检测体系的建立及应用 | |
| 引用本文： | 段丽萍,曹言勇,李会勇,陈洵,石磊.玉蜀黍黑粉菌环介导等温扩增检测体系的建立及应用[J].植物病理学报,2018,48(4):482-491. |
| 作者姓名： | 段丽萍 曹言勇 李会勇 陈洵 石磊 |
| 作者单位： | 暨南大学食品安全与营养研究院,广州510632; 河南省农业科学院粮食作物研究所,郑州450002 |
| 基金项目： | 国家重点研发计划项目(2016YFD0500600);河南省重点科技攻关项目(162102110003) |
| 摘 要： | 本文根据玉蜀黍黑粉菌(Ustilago maydis )的UmPep1、UmPit2和UmSee1基因各设计4套环介导等温扩增(LAMP)引物,从中筛选出1套引物对LAMP反应体系进行3因素(Bst DNA聚合酶浓度、Mg2+浓度、内外引物浓度比)3水平的优化试验。并对优化的U. maydis LAMP反应体系进行特异性、灵敏度及田间检测可行性试验。特异性试验表明,该方法能特异性检测U. maydis,而与其他病原菌的DNA没有交叉反应;灵敏度试验表明,该反应体系的最低检出限为44 fg·μL-1 pEasy-Pep质粒DNA,制作的标准曲线可对U. maydis进行定量分析。该方法也适用于在U. maydis侵染前或侵染早期对田间样品进行检测,对现场采集的172份田间样品进行检测,其中140个样品显示为阳性。本研究所建立的LAMP体系具有特异性好、灵敏度高、重复性好的特点,并能在45 min内完成对田间样品的检测,是快速、定量检测U. maydis的有效手段。 |
| 关 键 词： | 玉蜀黍黑粉菌 环介导等温扩增(LAMP) qPCR |
| 收稿时间： | 2017-08-24 |
Optimization and application of LAMP detection system for Ustilago maydis |
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| DUAN Li-ping,CAO Yan-yong,LI Hui-yong,CHEN Xun,SHI Lei.Optimization and application of LAMP detection system for Ustilago maydis[J].Acta Phytopathologica Sinica,2018,48(4):482-491. | |
| Authors: | DUAN Li-ping CAO Yan-yong LI Hui-yong CHEN Xun SHI Lei |
| Institution: | Institute of Food Safety and Nutrition, Jinan University, Guangzhou 510632,China; Institute of Cereal Crops,Henan Academy of Agricultural Sciences, Zhengzhou,450002,China |
| Abstract: | To screen the optimized target gene for LAMP assay, a total of 12 sets of LAMP primers, 4 sets for each gene, that targeted three Ustilago maydis effector genes, including UmPep1 , UmPit2 and UmSee1, were designed and screened. Out of the 12 setsprimer set Pep-2 was haphazardly selected to undergo three optimization experiments by combining a single factor experiment and an orthogonal design arrangement, such experiments include Mg2+, the optimal concentrations of Bst DNA polymerase, as well as internal/external primer ratio of the LAMP reaction system. After the optimization, specificity, sensitivity, and feasibility of application in the fields for LAMP assay were further analyzed. Based on the specificity analysis, this real-time LAMP (RealAmp) assay was confirmed by negative testing for other pathogens. The sensitivity analysis indicates that the detection sensitivity of the pathogen by the species-specific primer is as low as 44 fg·μL-1 pEasy-Pep plasmid DNA, and the standard curve obtained from the reaction exhibited a good linearity. For real-time feasibility of the application, results of the RealAmp assay for quantifying the genomic DNA of U. maydis were confirmed by testing with both artificially and naturally infected samples. Specifically, 140 samples out of 172 samples,100 soil and 72 maize plant samples,which were detected by LAMP using the ZYD-S1 system, tested positive. The successful sensitivity test of LAMP in artificially inoculated samples demonstrated that it might apply for the detection of field samples prior to or during the early stage of the U. maydis infection.To summarize, the LAMP method established in this study has proven to provide strong specificity and high sensitivity of the LAMP assay with in 45 minutes, which is also more efficient to monitor U. maydis in the field, as a simple, effective, and repeatable technique. |
| Keywords: | Ustilago maydis Loop-mediated isothermal amplification(LAMP) real-time polymerase chain reaction |
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