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作物学报 ›› 2025, Vol. 51 ›› Issue (5): 1248-1260.doi: 10.3724/SP.J.1006.2025.44113

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

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

殷丛丛1，李睿琦2，岳霈尧2，李晨1，牛景萍3，赵晋忠1，杜维俊2，岳爱琴2,*   

  1. 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
  • 收稿日期:2024-07-09 修回日期:2025-02-17 接受日期:2025-02-17 出版日期:2025-05-12 网络出版日期:2025-02-20
  • 基金资助:
    本研究由国家重点研发计划项目(2021YFD1600601-2), 山西省科技重大专项计划揭榜挂帅项目子课题(202201140601025-3-06), 山西省种业创新良种联合攻关子课题(YZGG-03-04-01)和山西省“1331”工程—作物学一流学科建设项目。

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

YIN Cong-Cong1,LI Rui-Qi2,YUE Pei-Yao2,LI Chen1,NIU Jing-Ping3,ZHAO Jin-Zhong1,DU Wei-Jun2,YUE Ai-Qin2,*   

  1. 1 Department of Basic Sciences, Shanxi Agricultural University, Jinzhong 030801, Shanxi, China; 2 College of Agronomy, Shanxi Agricultural University, Jinzhong 030801, Shanxi, China; 3 College of Life Sciences, Shanxi Agricultural University, Jinzhong 030801, Shanxi, China
  • Received:2024-07-09 Revised:2025-02-17 Accepted:2025-02-17 Published:2025-05-12 Published online:2025-02-20
  • Supported by:
    This study was supported by the National Key Research and Development Program of China (2021YFD1600601-2), the Sub-project of the Shanxi Province Science and Technology Major Special Plan Unveiling Project (202201140601025-3-06), the Sub-project of the Shanxi Province Seed Industry Innovation and Improved Seed Joint Research and Development Project (YZGG-03-04-01), and the Shanxi “1331 Project” Crop Science First-Class Discipline Construction Project.

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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(Closed dumbbell 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0.6 μmol 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10?4 ng μL?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%。

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

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 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%.

Key words: soybean mosaic virus, visual detection, closed dumbbell mediated isothermal amplification, nucleic acid detection, system optimization

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