| 侯雨萌1, 2, 赵振兴2, 王思元2, 董 铮2, 胡中泽3, 周 涛1*, 张永江2*.基于RT-RAA的苹果坏死花叶病毒快速检测方法的建立[J].植物保护,2025,51(4):314-319. |
| 基于RT-RAA的苹果坏死花叶病毒快速检测方法的建立 |
| Establishment of a rapid detection method for apple necrotic mosaic virus based on RT-RAA |
| 投稿时间：2024-09-23 修订日期：2024-12-18 |
| DOI：10.16688/j.zwbh.2024499 |
| 中文关键词: 苹果坏死花叶病毒(ApNMV) 反转录重组酶介导恒温扩增 快速检测 |
| 英文关键词:apple necrotic mosaic virus (ApNMV) reverse transcription-recombinase aided amplification rapid detection |
| 基金项目:国家重点研发计划（2023YFF0614402） |
| 作者 | 单位 | E-mail | | 侯雨萌1, 2, 赵振兴2, 王思元2, 董 铮2, 胡中泽3, 周 涛1*, 张永江2* | 1. 中国农业大学植物保护学院, 北京 100193
2. 中国检验检疫科学研究院, 北京 100176
3. 江苏省农业科学院泰州农科所, 泰州 225300 | 周涛taozhousig@163.com；张永江zhangyjpvi@yeah.net |
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| 中文摘要: |
| 苹果是我国重要的园艺作物, 具有较高的经济地位。病毒是引起苹果病害的重要因素, 苹果坏死花叶病毒(apple necrotic mosaic virus, ApNMV)是引起苹果坏死和花叶症状的病原之一。为了有效预防ApNMV的侵染, 本研究基于反转录重组酶介导恒温扩增(reverse transcription-recombinase aided amplification, RT-RAA)技术, 建立ApNMV的快速检测方法。根据ApNMV外壳蛋白编码基因的保守序列设计4对RAA引物, 筛选最佳引物序列, 并进行反应体系的优化。结果表明，RAA的最佳反应条件为:引物终浓度0.8 μmol/L, 反应温度42℃, 反应时间20 min。该方法能够特异性检测ApNMV。对携带ApNMV样品的RNA进行检测, RT-RAA检测体系的检测极限为5 pg/μL, 普通RT-PCR检测体系的检测极限为50 pg/μL, RT-RAA检测体系灵敏度是普通RT-PCR体系的10倍。该检测方法具有快速、简便、灵敏度高等优点。 |
| 英文摘要: |
| Apple is an important horticultural crop in China with high economic value. Viruses are among the primary pathogens affecting apple production, with apple necrotic mosaic virus (ApNMV) known to cause necrosis and mosaic symptoms. In order to effectively prevent the infection of ApNMV, a diagnostic method based on reverse transcription-recombinase aided amplification (RT-RAA) was developed in this study. Four pairs of RAA primers targeting the conserved regions of the ApNMV coat protein gene were designed, and the optimal pair was selected for further assay optimization. The final RT-RAA reaction conditions were set at a primer concentration of 0.8 μmol/L, a reaction temperature of 42℃ and a reaction time of 20 min. The assay showed high specificity for ApNMV, with a detection limit of of 5 pg/μL, compared to 50 pg/μL for conventional RT-PCR. Thus, the RT-RAA assay was approximately 10 times more sensitive than traditional RT-PCR. This method offers a rapid, simple, and highly sensitive approach for ApNMV detection, and holds promise for field diagnosis and quarantine applications. |
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