膜吸附法结合可视化环介导等温扩增技术检测柑橘黄龙病菌  

Detection of ’Candidatus Liberibacter asiaticus’ by Membrane Adsorption Method Combined with Visual Loop-Mediated Isothermal Amplification

作　　者：李镇希 李文婷 黄家权 郑正 许美容 邓晓玲[1] LI ZhenXi;LI WenTing;HUANG JiaQuan;ZHENG Zheng;XU MeiRong;DENG XiaoLing(College of Plant Protection,South China Agricultural University,Guangzhou 510642)

机构地区：[1]华南农业大学植物保护学院,广州510642

出　　处：《中国农业科学》2022年第1期74-84,共11页Scientia Agricultura Sinica

基　　金：国家重点研发计划(2018YFD0201500);广西创新驱动发展专项(桂科AA18118046);广东省重点领域研发计划(2019B020217003)。

摘　　要：【背景】柑橘黄龙病(Huanglongbing,HLB)是由候选韧皮部杆菌亚洲种(‘Candidatus Liberibacter asiaticus’,CLas)侵染引起的一种柑橘病害。对于该病害的防治主要采取综合措施,包括实施检疫、种植无病苗木、及时挖除病树和集中大面积联防联控柑橘木虱等。前3种方法都需要依靠准确的柑橘黄龙病诊断技术。【目的】利用环介导等温扩增技术(loop-mediated isothermal amplification, LAMP),结合膜吸附法DNA快速提取和Gelgreen荧光染料可视化,建立黄龙病田间核酸快速检测方法。【方法】以黄龙病菌β-操纵子与原噬菌体DNA聚合酶基因为模板设计LAMP特异性引物,包括外引物F3/B3、内引物FIP/BIP、环引物LoopF/LoopB和茎引物StemF/StemB。通过对环引物和茎引物设定不同的用量组合,对LAMP引物体系进行优化,确定合适的引物浓度。用优化后的LAMP引物体系对188份田间柑橘叶片进行检测,并构建受试者工作特征(receiver operating characteristic,ROC)曲线分析实时荧光LAMP(qLAMP)在CLas检测上的准确性。对qLAMP预混反应液进行两步的常温干燥,分别设定不同的存放条件,评估LAMP常温干燥试剂的稳定性。用本研究建立的CLas可视化LAMP快速检测方法,对田间71个柑橘叶片样品和35个柑橘果实样品进行检测,与实时荧光定量PCR(qPCR)比较两者的符合率。【结果】在LAMP体系中加入环引物、茎引物或增加其浓度都能促进反应速率的提升,并且同时加入终浓度均为1.6μmol·L-1的环引物和茎引物能进一步提高LAMP反应速率。LAMP预混液通过两步法干燥在不同温度存放1—4周均能保持反应活性基本不变,表明制备的两步法干燥LAMP试剂检测性能较好,在低温和常温环境下的稳定性尚可,仅35℃高温存放会略微增加LAMP试剂的反应时间。使用孔径0.1μm尼龙膜代替纤维素滤纸作为核酸吸附材料能提升CLas快速诊断技术的灵敏度。结合DNA快速提�【Background】Citrus Huanglongbing(HLB) is a citrus disease caused by ‘Candidatus Liberibacter asiaticus’(CLas). The main approaches to control HLB include plant quarantine, establishing disease-free nurseries, removing disease trees, and concentrating on large area joint control of citrus psyllids(Diaphorina citri). The first three methods all rely on accurate HLB diagnosis techniques.【Objective】The objective of this study is to establishment of a rapid and handy field/laboratory nucleic acid detection method of CLas using loop-mediated isothermal amplification(LAMP) combined with membrane adsorption rapid DNA extraction and Gelgreen fluorescence dye visualization.【Method】The LAMP primers were designed using the β-operon and the prophage DNA polymerase gene of CLas as templates, including outer primer F3/B3, inner primer FIP/BIP, loop primer LoopF/LoopB and stem primer StemF/StemB. The LAMP primer set was optimized by setting different dosage combinations for loop primers and stem primers to determine the appropriate primer concentration. A total of 188 field citrus leaves were detected using the optimized LAMP primer set, and the receiver operating characteristic(ROC) curves were constructed to analyze the accuracy of real-time fluorescent LAMP(qLAMP) for CLas detection. The qLAMP premixed reaction solution was dried in two steps at room temperature, storage temperature(4, 25 and 35℃) and storage time(1, 2 and 4 weeks) were also set to assess the enzyme activity stability of the dry LAMP reagent. Using dry LAMP reagent, combined with membrane adsorption rapid DNA extraction technique in this study, 71 citrus leaf samples and 35 citrus fruit samples collected in the field were detected, while the detection results of real-time fluorescent quantitative(qPCR) were used as controls to compare the coincidence rates of the two detection methods.【Result】The addition of loop primer, stem primer or increasing their concentrations in LAMP reaction could promote the increase of reaction rate, and the

关 键 词：柑橘黄龙病 候选韧皮部杆菌亚洲种 田间检测 膜吸附法 DNA快速提取 可视化环介导等温扩增技术 

分 类 号：S436.66[农业科学—农业昆虫与害虫防治]