苹果褪绿叶斑病毒RT-LAMP检测方法的建立  

Establishment of RT-LAMP Assay for Detection of Apple chlorotic leaf spot virus(ACLSV)

作　　者：张双纳 李正男[1] 范旭东[1] 张尊平[1] 任芳[1] 胡国君[1] 董雅凤[1] ZHANG ShuangNa;LI ZhengNan;FAN XuDong;ZHANG ZunPing;REN Fang;HU GuoJun;DONG YaFeng(National Center for Eliminating Viruses from Deciduous Fruit Tree, Institute of Pomology, Chinese Academy of Agricultural Sciences, Xingcheng 125100, Liaoning)

机构地区：[1]中国农业科学院果树研究所国家落叶果树脱毒中心,辽宁兴城125100

出　　处：《中国农业科学》2018年第9期1706-1716,共11页Scientia Agricultura Sinica

基　　金：中央级公益性科研院所基本科研业务费专项(1610182016017);中国农业科学院科技创新工程(ZX75-2018)

摘　　要：【目的】建立一种利用反转录环介导等温扩增(reverse transcription loop-mediated isothermal amplification,RT-LAMP)技术简便、快速检测苹果褪绿叶斑病毒(Apple chlorotic leaf spot virus,ACLSV)的方法。【方法】在ACLSV基因组序列的3个保守区域设计3组引物,每组引物包括一对外引物(F3/B3)和一对内引物(FIP/BIP),从3组引物中筛选出一组效果最好的引物。对RT-LAMP反应体系,即Mg^(2+)、d NTPs、Betaine、FIP/BIP、B3/F3浓度进行优化,Mg^(2+)浓度梯度设置为0、2.0、4.0、6.0、8.0、10.0、12.0 mmol·L^(-1),d NTPs浓度为0.6、0.8、1.0、1.2、1.4、1.6、1.8 mmol·L^(-1),Betaine浓度为0、0.2、0.4、0.6、0.8、1.0、1.2 mol·L^(-1),FIP/BIP浓度为0.8、1.2、1.6、2.0、2.4μmol·L^(-1),F3/B3浓度为0、0.1、0.2、0.3、0.4μmol·L^(-1);优化RT-LAMP反应条件,采用已优化的反应体系,设置65、63、61、59、57℃5个不同的反应温度,反应时间设定为90 min。在引物筛选和反应体系反应条件优化过程中,使用荧光定量PCR仪,在反应体系中加入荧光染料,利用荧光信号积累实时监测整个反应过程,根据扩增曲线判断反应结果。以携带苹果茎沟病毒(Apple stem grooving virus,ASGV)、苹果茎痘病毒(Apple stem pitting virus,ASPV)、苹果花叶病毒(Apple mosaic virus,Ap MV)的植株叶片中提取的总RNA为模板测试RT-LAMP检测方法的特异性。将含ACLSV的叶片总RNA原液进行10倍梯度稀释,以RNA原液和10^(-1)、10^(-2)、10^(-3)、10^(-4)、10^(-5)、10^(-6)稀释液作为模板,进行RT-LAMP反应,测试RT-LAMP检测方法的灵敏性。随机采集23株苹果树的叶片,同时进行RT-LAMP和RT-PCR检测,加入SYBR GreenⅠ进行可视化检测。【结果】建立了ACLSV RT-LAMP检测方法,优化的检测体系为:6.0 mmol·L^(-1) Mg^(2+)、1.2 mmol·L^(-1) d NTPs、0.2 mol·L^(-1) Betaine、1.6μmol·L^(-1) FIP/BIP和0.2μmol·L^(-1) F3/B3引物,最佳反应条件为59℃,60 min。特异性检测中,仅ACLSV检测结果为�【Objective】The objective of this study is to establish a method which uses reverse transcription loop-mediated isothermal amplification（RT-LAMP） technology, and to detect Apple chlorotic leaf spot virus（ACLSV） simply and quickly. 【Method】Three sets of specific primers were designed based on conserved region of ACLSV genomes. Each set of primers includes a pair of outer primer（F3/B3） and a pair of inner primer（FIP/BIP）. One feasible set of primers was selected for the RT-LAMP reaction. RT-LAMP reaction system was optimized, that is, the concentration of Mg2＋（0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0 mmol·L^-1）, d NTPs（0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 mmol·L^-1）, Betaine（0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mol·L^-1）, FIP/BIP（0.8, 1.2, 1.6, 2.0, 2.4 μmol·L^-1） and F3/B3（0, 0.1, 0.2, 0.3, 0.4 μmol·L^-1）, respectively. The RT-LAMP reaction condition was optimized, using optimized reaction system, reaction temperature was set to 65, 63, 61, 59, 57℃ and reaction time was set for 90 min. In the process of primers screening and optimizing the reaction system, fluorescent pigment was added to the reaction system and real-time PCR instrument was used. The whole process was detected in real-time by fluorescence signal accumulation. Finally, the amplification curve was used to analyze the reaction result. The specificity detection of RT-LAMP was tested by using different RNA templates of infected leaves from ACLSV, Apple stem grooving virus（ASGV）, Apple stem pitting virus（ASPV） and Apple mosaic virus（Ap MV）. To assess the detection sensitivity, 100, 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6 diluents of original RNA were used as templates of RT-LAMP. To evaluate the application value of this method, 23 apple leaf samples were collected randomly in the field, the optimized RT-LAMP and RT-PCR were used to detect the samples, SYBR GreenⅠwas added to visualize the detection. 【Result】 The RT-LAMP method to detect ACLSV was established. The optimized detection system was 6

关 键 词：苹果 苹果褪绿叶斑病毒 反转录环介导等温扩增 REAL-TIME PCR 病毒检测 

分 类 号：S432.41[农业科学—植物病理学]