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芋疫霉重组聚合酶扩增结合侧流层析试纸条快速检测方法的建立及应用

引用本文：王荣波,陈姝樽,赵玉梅,李本金,刘裴清,陈庆河.芋疫霉重组聚合酶扩增结合侧流层析试纸条快速检测方法的建立及应用.植物保护学报,2022,49(6):1654-1662

DOI：10.13802/j.cnki.zwbhxb.2021.2021178

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作者	单位	E-mail
王荣波	福建省农业科学院植物保护研究所, 福建省作物有害生物监测与治理重点实验室, 福州 350013 福建农林大学植物保护学院, 海峡两岸特色作物安全生产省部共建协同创新中心, 福州 350002
陈姝樽	福建省农业科学院植物保护研究所, 福建省作物有害生物监测与治理重点实验室, 福州 350013 福建农林大学植物保护学院, 海峡两岸特色作物安全生产省部共建协同创新中心, 福州 350002
赵玉梅	福建省农业科学院植物保护研究所, 福建省作物有害生物监测与治理重点实验室, 福州 350013 福建农林大学植物保护学院, 海峡两岸特色作物安全生产省部共建协同创新中心, 福州 350002
李本金	福建省农业科学院植物保护研究所, 福建省作物有害生物监测与治理重点实验室, 福州 350013 福建农林大学植物保护学院, 海峡两岸特色作物安全生产省部共建协同创新中心, 福州 350002
刘裴清	福建省农业科学院植物保护研究所, 福建省作物有害生物监测与治理重点实验室, 福州 350013 福建农林大学植物保护学院, 海峡两岸特色作物安全生产省部共建协同创新中心, 福州 350002
陈庆河	福建省农业科学院植物保护研究所, 福建省作物有害生物监测与治理重点实验室, 福州 350013 海南大学植物保护学院, 热带农林生物灾害绿色防控教育部重点实验室, 海口 570228	qhchen@hainanu.edu.cn

中文摘要:为建立芋疫霉Phytophthora colocasiae快速准确的分子检测方法，基于Ypt1基因特异序列，设计芋疫霉的特异性引物与探针，建立一种快速、准确、可视化的芋疫霉重组聚合酶扩增结合侧流层析试纸条（recombinase polymerase amplification-lateral flow dipstick，LFD-RPA）检测方法，对该检测方法进行优化，评估其特异性与灵敏度，并对田间疑似样品进行检测。结果表明，优化后的芋疫霉LFD-RPA检测方法最适反应条件为39℃恒温反应30 min。LFD-RPA检测方法能够特异性地检测出芋疫霉，而对其他卵菌近缘种和常见植物病原真菌均未检出，且该检测方法对芋疫霉DNA的检测灵敏度达到1 pg/μL。对田间带病组织检测发现，LFD-RPA检测方法能够快速准确地从田间自然发病植株中检测出芋疫霉。表明本研究所建立的芋疫霉LFD-RPA快速可视化检测方法特异性好、灵敏度高、简单快捷，可用于芋疫病的田间快速诊断。

中文关键词:芋疫霉重组聚合酶扩增结合侧流层析试纸条 Ypt1基因快速检测

Development of a recombinase polymerase amplification-lateral flow dipstick assay for rapid detection of the taro leaf blight pathogen Phytophthora colocasiae

Author Name	Affiliation	E-mail
Wang Rongbo	Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, Institute of Plant Protection, FujianAcademy of Agricultural Sciences, Fuzhou 350013, Fujian Province, China Ministerial and Provincial Joint Innovation Centrefor Safety Production of Cross-Strait Crops, College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China
Chen Shuzun	Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, Institute of Plant Protection, FujianAcademy of Agricultural Sciences, Fuzhou 350013, Fujian Province, China Ministerial and Provincial Joint Innovation Centrefor Safety Production of Cross-Strait Crops, College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China
Zhao Yumei	Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, Institute of Plant Protection, FujianAcademy of Agricultural Sciences, Fuzhou 350013, Fujian Province, China Ministerial and Provincial Joint Innovation Centrefor Safety Production of Cross-Strait Crops, College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China
Li Benjin	Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, Institute of Plant Protection, FujianAcademy of Agricultural Sciences, Fuzhou 350013, Fujian Province, China Ministerial and Provincial Joint Innovation Centrefor Safety Production of Cross-Strait Crops, College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China
Liu Peiqing	Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, Institute of Plant Protection, FujianAcademy of Agricultural Sciences, Fuzhou 350013, Fujian Province, China Ministerial and Provincial Joint Innovation Centrefor Safety Production of Cross-Strait Crops, College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China
Chen Qinghe	Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, Institute of Plant Protection, FujianAcademy of Agricultural Sciences, Fuzhou 350013, Fujian Province, China Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests, Ministry of Education, College of Plant Protection, Hainan University, Haikou 570228, Hainan Province, China	qhchen@hainanu.edu.cn

Abstract:To develop a rapid and accurate molecular detection method for the taro leaf blight pathogen Phytophthora colocasiae, recombinase polymerase amplification combined with a lateral flow dipstick (LFD-RPA) assay was established for sensitive detection of P. colocasiae. Specific primers and probe for P. colocasiae were designed targeting ras-related protein gene Ypt1. The reaction conditions of LFDRPA assay were optimized, the specificity and sensitivity of the assay were tested, and the optimized procedure was evaluated for detection of pathogen in inoculated plant tissues. The optimized reaction conditions of LFD-RPA assay were a constant temperature of 39℃ and an incubation time of 30 min. The specificity test showed that LFD-RPA assay was specific to P. colocasiae, and yielded negative results for other related oomycetes and fungal species. The detection limit of LFD-RPA assay was 1 pg/μL of genomic DNA. Furthermore, LFD-RPA assay quickly and accurately detected P. colocasiae from naturally infected leaves of taro in the field. Overall, the LFD-RPA assay developed in this study is a rapid, simple, and sensitive method for detecting P. colocasiae, and has the potential for diagnosing taro leaf blight in the fields.

keywords:Phytophthora colocasiae recombinase polymerase amplification-lateral flow dipstick (LFDRPA) Ypt1 gene rapid diagnosis

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