作物学报2025，Vol.51Issue(5)：1248-1260,中插8-中插9,15.DOI:10.3724/SP.J.1006.2025.44113
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
摘要
Abstract
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 0.6 μmol 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 ng μL-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Key words
soybean mosaic virus/visual detection/closed dumbbell mediated isothermal amplification/nucleic acid detection/system optimization引用本文复制引用
殷丛丛,李睿琦,岳霈尧,李晨,牛景萍,赵晋忠,杜维俊,岳爱琴..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[J].作物学报,2025,51(5):1248-1260,中插8-中插9,15.基金项目
本研究由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)和山西省"1331"工程—作物学一流学科建设项目资助. This study was supported by the Sub-project of the Shanxi Province Science and Technology Major Special Plan Unveiling Project(202201140601025-3-06),Sub-project on Key Core Technology Breakthroughs in Agriculture of Shanxi Province(NYGG27-04-01),and the Shanxi"1331 Project"Crop Science First-Class Discipline Construction Project. （202201140601025-3-06）
