余甘子果实斑点病菌LAMP可视化检测方法的建立  

Establishment of loop-mediated isothermal amplification(LAMP)for visual rapid detection of Diaporthe phoenicicola of fruit spot disease on Phyllanthus emblica

作　　者：赖多[1] 王德林 邵雪花[1] 秦健 庄庆礼 肖维强[1] LAI Duo;WANG Delin;SHAO Xuehua;QIN Jian;ZHUANG Qingli;XIAO Weiqiang(Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization,Ministry of Agriculture and Rural Affairs/Guangdong Provincial Key Laboratory of Science and Technology Research on Fruit Tree,Institute of Fruit Tree Research,Guangdong Academy of Agricultural Sciences,Guangzhou,Guangdong 510640,China)

机构地区：[1]广东省农业科学院果树研究所农业农村部南亚热带果树生物学与遗传资源利用重点实验室/广东省果树科学与技术研究重点实验室,广东广州510640

出　　处：《西北农林科技大学学报(自然科学版)》2025年第1期69-79,90,共12页Journal of Northwest A&F University(Natural Science Edition)

基　　金：广东省农业科学院汕尾分院科技合作专项(2023分院专项01);汕尾市科技计划项目(2023B006);广东省农业农村厅农业科研类及技术推广示范类项目(2022)。

摘　　要：【目的】建立余甘子果实斑点病菌快速、便捷、灵敏、可视的环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)检测方法,为该病害的风险预判和科学防治提供依据。【方法】以余甘子果实斑点病致病菌间座壳菌(Diaporthe phoenicicola)的翻译延伸因子EF-1α基因为靶序列,设计LAMP特异性扩增引物;以余甘子间座壳菌DNA为模板,优化反应温度、反应时间、dNTPs浓度、Mg^(2+)浓度和内外侧引物浓度比,建立LAMP检测最佳反应体系,测试其特异性和灵敏性,并对余甘子发病果实进行实际检测。【结果】适用于余甘子果实斑点病LAMP检测的最佳引物为引物组合4(EF4-F3/EF4-B3和EF4-FIP/EF4-BIP);LAMP检测最佳反应条件为dNTPs浓度1.0 mmol/L,Mg^(2+)浓度4 mmol/L,内外侧引物质量比6∶1,反应温度63℃,反应时间50 min。优化后的LAMP检测方法可特异性地检出余甘子间座壳菌(D.phoenicicola),检测灵敏度可达0.01 ng/μL,且在实际应用中准确率高达100%。【结论】首次建立了余甘子果实斑点病致病菌间座壳菌LAMP可视化快速检测方法,该方法特异性强、灵敏度高,操作简单,可用于田间余甘子果实斑点病菌的快速检测。【Objective】This study is to establish a rapid,convenient,and sensitive loop-mediated isothermal amplification(LAMP)method for visual detection of Diaporthe phoenicicola that causes fruit spot disease on Phyllanthus emblica,and to provide theoretical basis for the risk prediction and disease control.【Method】The specific amplification primers for LAMP were designed with the translation extension factor gene EF-1αof D.phoenicicola as the target sequence.With D.phoenicicola DNA as the template,the LAMP detection reaction system was optimized by adjusting the reaction temperature,reaction time,concentration of dNTPs,concentration of Mg^(2+)and concentration ratio of inner and outer primers.The specificity and sensitivity of the system were tested,and practical detection was conducted on the di-seased fruits of P.emblica.【Result】Primer set 4(EF4-F3/EF4-B3和EF4-FIP/EF4-BI)was the best primer pair for LAMP detection of fruit spot disease of P.emblica.The optimal reaction conditions of LAMP test were dNTPs concentration of 1.0 mmol/L,Mg^(2+)concentration of 4 mmol/L,inner to outer primer concentration ratio of 6∶1,reaction temperature of 63℃,and reaction time of 50 min.The optimized LAMP detection method could specifically detect D.phoenicicola,with a sensitivity of 0.01 ng/μL,and an accuracy rate of 100%in practical application.【Conclusion】In this study,LAMP visual rapid detection method for D.phoenicicola on P.emblica fruit was established for the first time,with a high specificity,high sensitivity,and great simplity in its operation,which can be used for rapid detection in the field.

关 键 词：余甘子 间座壳菌 环介导等温扩增技术 灵敏性 特异性 可视化 

分 类 号：S435.79[农业科学—农业昆虫与害虫防治]