基于闭合哑铃介导等温扩增可视化检测大豆花叶病毒SC15方法的建立及应用  

Establishment and application of a visual detection method for soybean mosaic virus SC15 based on closed dumbbell mediated isothermal amplification

作　　者：殷丛丛 李睿琦 岳霈尧 李晨[1] 牛景萍 赵晋忠[1] 杜维俊[2] 岳爱琴[2] YIN Cong-Cong;LI Rui-Qi;YUE Pei-Yao;LI Chen;NIU Jing-Ping;ZHAO Jin-Zhong;DU Wei-Jun;YUE Ai-Qin(Department of Basic Sciences,Shanxi Agricultural University,Jinzhong 030801,Shanxi,China;College of Agronomy,Shanxi Agricultural University,Jinzhong 030801,Shanxi,China;College of Life Sciences,Shanxi Agricultural University,Jinzhong 030801,Shanxi,China)

机构地区：[1]山西农业大学基础部,山西晋中030801 [2]山西农业大学农学院,山西晋中030801 [3]山西农业大学生命科学学院,山西晋中030801

出　　处：《作物学报》2025年第5期1248-1260,共13页Acta Agronomica Sinica

基　　金：山西省科技重大专项计划揭榜挂帅项目子课题(202201140601025-3-06);山西省农业关键核心技术攻关子课题(NYGG27-04-01);山西省“1331”工程—作物学一流学科建设项目资助。

摘　　要：大豆花叶病是一种由大豆花叶病毒(soybean mosaic virus,SMV)引起的最为普遍和严重的全球性大豆病害,可导致大豆产量和种子品质大幅降低,我国大豆产区均受其影响。在我国, SMV被划分为22个株系(SC1~SC22),其中SMV-SC15毒性最强。但是,目前尚无有效的早期诊断方法,本研究基于闭合哑铃介导等温扩增(closeddumbbell mediated isothermal amplification, CDA),建立了一种可视化快速检测SMV-SC15的方法,实现了对SC15的高效特异检测与鉴定。根据SMV不同株系CP基因组序列的多态性设计了CDA方法的引物对(MF/MR),建立并优化了检测SMV-SC15的反应体系,确定了最佳反应条件:反应温度63℃、Bst DNA聚合酶用量4.8 U以及引物浓度0.6μmol L^(-1)。以溴百里酚蓝(BTB)和SYBR Green Ⅰ为指示剂实现了检测结果的可视化。对比分析CDA体系和加环引物CDA体系(L-CDA)检测SMV-SC15的稳定性、特异性和灵敏度发现, L-CDA体系实时荧光扩增曲线达到阈值的时间比CDA缩短5~6 min,其最低检出浓度低至1×10^(–4) ngμL^(-1),灵敏度为CDA体系的10倍。本研究通过L-CDA体系检测了200份不同品种的田间大豆叶片样本,显色结果对应于RT-qPCR检测的Ct值约为32,其灵敏度和特异性分别为100%和96.3%。Soybean mosaic virus(SMV)disease is one of the most widespread and serious global soybean diseases,which can cause significant reductions in soybean yields and seed quality,and affect all soybean-producing regions in China.SMV is divided into 22 strains(SC1-SC22)in China,among which SMV-SC15 has stronger toxicity.However,there is currently no effective early diagnostic method.In this study,a visual and rapid method for detecting SMV-SC15 was established based on closed dumbbell mediated isothermal amplification(CDA),achieving highly specific and sensitive detection of SC15.Primer pairs(MF/MR)for CDA method was designed based on the polymorphism of CP sequences in different strains of SMV,and the reaction system for detecting SMV-SC15 was optimized.The optimal reaction conditions were obtained as follows:reaction temperature was 63℃,the dose of Bst DNA polymerase was 4.8 U,and primer concentration was 0.6μmol L^(-1).The detection results were visualized by using BTB and SYBR Green I as color developing agents.A comparative analysis was conducted on the stability,specificity and sensitivity of the CDA system and the CDA system with ring primers(L-CDA)for detecting SMV-SC15.The results showed that the time for real-time fluorescence amplification curve of L-CDA system to reach the threshold was 5-6 min shorter than that of CDA system,and the lowest detection limit was 1×10^(-4) ngμL^(-1),which was 10 times lower than that of CDA system.The L-CDA system was used to detect 200 soybean leaf samples of different varieties in the field,and the colorimetric result corresponds to a Ct value of approximately 32 in RT-qPCR detection,with a sensitivity of 100%and a specificity of 96.3%.

关 键 词：大豆花叶病毒 可视化检测 闭合哑铃介导等温扩增 核酸检测 体系优化 

分 类 号：S435.651[农业科学—农业昆虫与害虫防治]