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| 基于核酸适配体TNA1c构建靶标激活式探针用于特异性检测石斑鱼神经坏死病毒的感染 |
| A target activatable aptamer probe for specific detection of grouper nervous necrosis virus infection |
| 投稿时间：2025-02-21 修订日期：2025-04-18 |
| DOI： |
| 中文关键词: 石斑鱼 神经坏死病毒 快速检测技术 核酸适配体 靶标激活式探针 |
| 英文关键词: Grouper Nervous necrosis virus Rapid detection assay Aptamer Target activatable probe |
| 基金项目:国家自然科学基金项目 |
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| 摘要点击次数: 110 |
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| 中文摘要: |
| 石斑鱼神经坏死病毒(Nervous necrosis virus, NNV)作为石斑鱼养殖中最具破坏力的病毒性病原之一，对石斑鱼养殖业造成严重威胁，其引发的病毒性神经坏死病(Viral Nervous Necrosis, VNN)会导致仔鱼和幼鱼大规模死亡。因此，必须开发具备现场快速响应能力、操作便捷且兼具高灵敏度和特异性的新型检测技术，满足NNV早期诊断和精准防控的迫切需求。核酸适配体TNA1能够高特异性识别NNV感染细胞，本研究通过对TNA1进行结构改造和化学修饰，创新性地构建出一种靶标激活式核酸适配体探针(Target activated aptamer TNA1c, TNA1c-TAA)。利用流式细胞术、激光共聚焦技术以及实时荧光定量PCR技术，对TNA1c-TAA的特异性、灵敏性、稳定性进行分析，结果发现，TNA1c-TAA能够高特异性地识别NNV感染的细胞，不识别其它病毒感染的细胞。TNA1c-TAA (500 nM) 在4 - 28 ℃的温度范围内，能够在1 min内精准检测到1 × 103 个NNV感染的细胞。活体水平的检测结果显示，TNA1c-TAA可以特异性识别NNV感染的石斑鱼脑组织细胞，TNA1c-TAA检测样品的阳性率与qPCR检测结果一致，证实TNA1c-TAA具有应用于石斑鱼养殖中NNV感染快速诊断的潜力，为构建水产疫病“预防-诊断-控制”三位一体防控体系奠定理论基础。 |
| 英文摘要: |
| Nervous necrosis virus (NNV), one of the most devastating viral pathogens in grouper aquaculture, causes severe impacts on the grouper farming industry. NNV-induced viral nervous necrosis (VNN) triggers mass mortality in larvae and juveniles. There is an urgent need to develop novel detection technologies with on-site rapid response capability, operational simplicity, and high sensitivity/specificity to meet the demands for early diagnosis and precise prevention?. In this study, we innovatively constructed a target-activated aptamer probe (TNA1c-TAA) through structural modification and chemical engineering of TNA1, an aptamer capable of specifically recognizing NNV-infected cells?. Systematic evaluations using flow cytometry, confocal microscopy, and real-time quantitative PCR (qPCR) revealed that，TNA1c-TAA exhibited high specificity for NNV-infected cells without cross-reactivity to other virus-infected cells?. The TNA1c-TAA probe (500 nM) achieved precise detection of 1×103 NNV-infected cells within 1 minute across 4-28℃?. In vivo detection demonstrated specific recognition of NNV-infected brain tissue cells in grouper, with 100% concordance between TNA1c-TAA positivity rate and qPCR results?. The findings validate TNA1c-TAA's potential for rapid diagnosis of NNV disease in aquaculture, laying the foundation for establishing a trinity prevention-diagnosis-control system against aquatic epidemics?. |
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