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  

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

作　　者：王荣波[1,2] 陈姝樽 赵玉梅 李本金 刘裴清[1,2] 陈庆河 Wang Rongbo;Chen Shuzun;Zhao Yumei;Li Benjin;Liu Peiqing;Chen Qinghe(Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests,Institute of Plant Protection,Fujian Academy of Agricultural Sciences,Fuzhou 350013,Fujian Province,China;Ministerial and Provincial Joint Innovation Centre for Safety Production of Cross-Strait Crops,College of Plant Protection,Fujian Agriculture and Forestry University,Fuzhou 350002,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)

机构地区：[1]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 [2]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 [3]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

出　　处：《植物保护学报》2022年第6期1654-1662,共9页Journal of Plant Protection

基　　金：国家自然科学基金(31801683,31772141);福建省农业科学院对外合作项目(DWHZ2021-13);5511协同创新工程(XTCXGC2021011,XTCXGC2021017)。

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。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.

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 

分 类 号：S436.32[农业科学—农业昆虫与害虫防治]