基于环介导等温扩增技术检测烟草疫霉菌  

Detection of Phytophthora nicotianae based on a LAMP technique

作　　者：朱百佳 刘晓宇 李昕建 韦兴启 罗有江 刘垦 游安弟 雷庭 王杰[2] 罗倩茜 ZHU Baijia;LIU Xiaoyu;LI Xinjian;WEI Xingqi;LUO Youjiang;LIU Ken;YOU Andi;LEI Ting;WANG Jie;LUO Qianxi(Guizhou Tobacco Research Institute,Guiyang,Guizhou 550000,China;Institute of Tobacco,Chinese Academy of Agricultural Sciences,Qingdao,Shandong 266101,China)

机构地区：[1]贵州省烟草科学研究院,贵州贵阳50000 [2]中国农业科学院烟草研究所,山东青岛266101

出　　处：《生物灾害科学》2025年第1期82-91,共10页Biological Disaster Science

基　　金：贵州省烟草公司科技项目(2023522700240236)。

摘　　要：【目的】应用环介导等温核酸扩增技术(loop-mediated isothermalamplification,LAMP)建立基于颜色判定的简单、快速、灵敏并具有通用性的烟草疫霉(Phytophthora parasitica var. nicotianae)检测方法,为该病害的风险预判和防治提供依据。【方法】以PUF蛋白编码基因PpPuf4为靶序列,设计并筛选得到一组烟草疫霉菌特异性LAMP引物,建立了一种快速有效的LAMP检测体系。通过实验对该检测方法的特异性、灵敏度、通用性进行了评估,并对其实际应用效果进行了验证。【结果】在62℃恒温条件下进行扩增反应,反应结束后加入荧光染料SUPERGreenI作为显色剂,特异性检测结果显示,只有烟草疫霉菌株的LAMP反应液为阳性(黄绿色),另外14株其他烟草常见真菌病原菌反应液为阴性。灵敏度试验结果显示,最低能检测到烟草疫霉的DNA浓度为1 pg/μL。通用性结果显示18株不同地区来源的烟草疫霉菌均可被检出。人工接种发病组织和田间黑胫病烟株的检测结果均呈阳性,与组织分离法对烟草疫霉的检测结果相一致。【结论】该检测方法的建立,可用于田间烟草黑胫病的快速、简单、有效地检测,有利于烟草黑胫病的及时发现和防控。[Objective]Loop-mediated isothermal amplification(LAMP)was used to establish a simple,rapid,sensitive,and universal method for the detection of Phytophthora nicotianae based on color determination for disease risk prediction and control.[Method]A set of Phytophthora nicotianae-specific LAMP primers was designed and screened with the PUF protein-coding gene PpPuf4 as the target sequence,and a rapid and effective LAMP detection system was established.The specificity,sensitivity,and versatility of the assay were also evaluated experimentally,and its practical application was verified.[Result]The amplification reaction was carried out at a constant temperature of 62℃,and the fluorescent dye SUPER Green I was added as a color developer at the end of the reaction.The results of the specificity test showed that only the Phytophthora nicotianae strains of the LAMP reactants were positive(yellow-green),and the reactants of 14 strains of the other common fungal pathogens of tobacco were negative.The sensitivity test showed that the lowest detectable Phytophthora nicotianae DNA concentration was 1 pg/μL.The generalisability results showed that 18 strains of Phytophthora nicotianae from different regional sources could be detected.The results of artificial inoculation of diseased tissues and field black shank tobacco plants were positive,consistent with the tissue isolation method for Phytophthora nicotianae.[Conclusion]The development of this detection method allows for rapid,simple,and effective identification of tobacco black shank disease in the field,thereby facilitating timely detection and management of the disease.

关 键 词：环介导等温扩增 烟草疫霉 烟草黑胫病 SUPER GreenⅠ 检测技术 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]