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| 基于致病基因序列的LAMP方法检测番茄种子携带的溃疡病菌 | |
| 引用本文： | 吕青阳,蒋 娜,韩思宁,李健强,罗来鑫.基于致病基因序列的LAMP方法检测番茄种子携带的溃疡病菌[J].植物病理学报,2017,47(5):630-639. |
| 作者姓名： | 吕青阳 蒋 娜 韩思宁 李健强 罗来鑫 |
| 作者单位： | 中国农业大学植物病理学系,种子病害检验与防控北京市重点实验室,北京 100193 |
| 基金项目： | 国家自然科学基金项目(31571972) |
| 摘 要： | 本研究选取番茄溃疡病菌(Clavibacter michiganensis subsp.michiganensis,Cmm)致病岛上的chpC基因的部分序列,作为环介导等温扩增(loop-mediated isothermal amplification,LAMP)靶标片段进行LAMP引物设计。对反应体系优化后进行特异性测定,结果表明供试的89株番茄溃疡病菌中86株检测结果为阳性,3株为阴性,供试的14株非番茄溃疡病菌(其他重要植物病原细菌)均为阴性。检测番茄溃疡病菌菌悬液样品的阈值为4.8×10~5 CFU·mL~(-1),对DNA样品的检测阈值为1.8×10~(-2) ng·μL~(-1),并据此建立了番茄溃疡病菌的LAMP检测方法。将该方法应用于番茄种子携带Cmm的检测,通过提取种子浸提液样品的总DNA,实现了对番茄种子携带Cmm的直接检测。与普通PCR相比,该方法更加快捷简便,不依赖PCR仪等昂贵的仪器设备,可以丰富现有的番茄溃疡病菌分子检测体系,为口岸等检疫部门提供简单易行的检测初筛手段。 |
| 关 键 词： | 番茄溃疡病菌 环介导等温扩增 种子提取液 快速检测 |
Detection of Clavibacter michiganensis subsp. michiganensis in tomato seed by loop-mediated isothermal amplification method based on virulence gene sequence |
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LV Qing-yang,JIANG Na,HAN Si-ning,LI Jian-qiang,LUO Lai-xin.Detection of Clavibacter michiganensis subsp. michiganensis in tomato seed by loop-mediated isothermal amplification method based on virulence gene sequence[J].Acta Phytopathologica Sinica,2017,47(5):630-639. |
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| Authors: | LV Qing-yang JIANG Na HAN Si-ning LI Jian-qiang LUO Lai-xin |
| Institution: | Department of Plant Pathology, China Agricultural University; Beijing Key Laboratory of Seed Disease Testing and Control, Beijing 100193, China |
| Abstract: | The loop-mediated isothermal amplification (LAMP) method was used to detect Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato seeds. The LAMP primers were designed based on the virulence-related chpC gene sequence. Among 89 Cmm strains, 86 were detected as positive, and 14 non-Cmm plant pathogenic bacteria were detected as negative by LAMP method. The sensitivity were 4.3×105 CFU·mL-1 for Cmm suspensions and 1.8×10-2 ng·μL-1 for Cmm DNA, respectively. These results indicated the high specificity and sensitivity of the primers and the LAMP system can be used in detecting Cmm from tomato seed by directly isolating DNA from seed extract. The results showed that LAMP is more rapid and simple than general PCR for detecting Cmm from tomato seeds. The LAMP method is cost-effective and can be simply performed in water bath rather than in PCR thermal cycler. Therefore, the LAMP assay provides a rapid prescreen for tomato seed health to enhance the efficiency of Cmm detection in the inspection stations at the ports of entry. |
| Keywords: | Clavibacter michiganensis subsp michiganensis loop-mediated isothermal amplification seed extract rapid detection |
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