利用环介导等温扩增方法快速检测瓜类细菌性果斑病菌和番茄细菌性溃疡病菌 |
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引用本文:吴秀芹,杨樱子,罗金燕,邱文,安千里,李斌.利用环介导等温扩增方法快速检测瓜类细菌性果斑病菌和番茄细菌性溃疡病菌.植物保护学报,2018,45(6):1235-1241 |
DOI:10.13802/j.cnki.zwbhxb.2018.2017106 |
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作者 | 单位 | E-mail | 吴秀芹 | 浙江大学生物技术研究所, 农业部作物病虫分子生物学重点实验室, 杭州 310058 | | 杨樱子 | 浙江大学生物技术研究所, 农业部作物病虫分子生物学重点实验室, 杭州 310058 | | 罗金燕 | 上海市农业技术推广服务中心, 上海 201103 | | 邱文 | 浙江大学生物技术研究所, 农业部作物病虫分子生物学重点实验室, 杭州 310058 | wqiu2015@zju.edu.cn | 安千里 | 浙江大学生物技术研究所, 农业部作物病虫分子生物学重点实验室, 杭州 310058 | | 李斌 | 浙江大学生物技术研究所, 农业部作物病虫分子生物学重点实验室, 杭州 310058 | libin0571@zju.edu.cn |
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中文摘要:为建立简单、快速和灵敏地检测瓜类细菌性果斑病菌Acidovorax avenae subsp.citrulli(Aac)和番茄细菌性溃疡病菌Clavibacter michiganensis subsp.michiganensis(Cmm)的环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法,以Aac的ugpB基因和Cmm的micA基因为靶标,分别设计、合成和筛选特异性引物,摸索和优化各项反应条件和反应体系,成功建立了以钙黄绿素颜色为指示且只需要金属浴恒温反应30~60 min的LAMP扩增体系。特异性分析表明该LAMP方法可以快速检出5株不同的Aac菌株和2株不同的Cmm菌株,其它对照菌株如燕麦嗜酸菌燕麦亚种A.avenae subsp.avenae、丁香假单胞菌Pseudomonas syringae、水稻黄单胞菌水稻致病变种Xanthomonas oryzae pv.oryzae和茄科雷尔氏菌Ralstonia solanacearum则呈现阴性反应;引物比目前报道的LAMP引物有更高的DNA样品检测灵敏度,Aac和Cmm的灵敏度分别为1.72×102 fg/μL和1.26×102 fg/μL。研究结果表明,所设计的Aac和Cmm引物特异性好、灵敏度高,更有利于从源头上控制这2种检疫性细菌病害的流行和传播。 |
中文关键词:瓜类细菌性果斑病菌 番茄细菌性溃疡病菌 环介导等温扩增方法 快速检测 |
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Rapid detection of Acidovorax avenae subsp. citrulli and Clavibacter michiganensis subsp. michiganensis with loop-mediated isothermal amplification |
Author Name | Affiliation | E-mail | Wu Xiuqin | Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture Institute of Biotechnology, Zhejiang University, Hangzhou 310058, Zhejiang Province, China | | Yang Yingzi | Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture Institute of Biotechnology, Zhejiang University, Hangzhou 310058, Zhejiang Province, China | | Luo Jinyan | Shanghai Agricultural Technology Extension & Service Center, Shanghai 201103, China | | Qiu Wen | Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture Institute of Biotechnology, Zhejiang University, Hangzhou 310058, Zhejiang Province, China | wqiu2015@zju.edu.cn | An Qianli | Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture Institute of Biotechnology, Zhejiang University, Hangzhou 310058, Zhejiang Province, China | | Li Bin | Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture Institute of Biotechnology, Zhejiang University, Hangzhou 310058, Zhejiang Province, China | libin0571@zju.edu.cn |
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Abstract:To establish a simple, sensitive and rapid loop-mediated isothermal amplification (LAMP) method for the detection of Acidovorax avenae subsp. citrulli (Aac) causing melon bacterial fruit blotch and Clavibacter michiganensis subsp. michiganensis (Cmm) causing tomato bacterial canker disease, ugpB gene of Aac and micA gene of Cmm were used as the target for design of LAMP primers. Following primers screening and optimization of reaction systems and conditions, the LAMP method was finally established based on 30-60 min incubation by using calcein as an indicator. It showed a good prospect for application due to the reduction in both the reaction time and cost. The results of specificity analysis indicated a positive reaction for the five strains of Aac and two strains of Cmm and a negative reaction for the reference strains A. avenae subsp. avenae, Pseudomonas syringae, Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, etc. In addition, the sensitivity of LAMP was 1.72×102 fg/μL for Aac and 1.26×102 fg/μL for Cmm, respectively, which were higher than those of the previous reports. In general, the LAMP system is highly sensitive and specific and helpful for control of the epidemics and spread of the two bacterial diseases. |
keywords:Acidovorax avenae subsp.citrulli Clavibacter michiganensis subsp.michiganensis loopmediated isothermal amplification technique rapid detection |
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