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水产品中GⅡ型诺如病毒可视化LAMP法的建立及应用
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1.上海海洋大学食品学院，上海 201306;2.上海海关动植物与食品检验检疫技术中心，上海 200135;3.上海水产品加工及贮藏工程技术研究中心，上海 201306;4.农业农村部水产品贮藏保鲜质量安全风险评估实验室(上海)，上海 201306;5.上海海洋大学食品热加工工程技术研究中心，上海 201306

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通讯作者:

刘海泉（1980—），男，博士，副教授，硕士研究生导师，主要从事食源性致病菌的快速检测。E-mail：hqliu@shou.edu.cn

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上海市“科技创新行动计划”农业科技领域项目（22N31900600）。


Establishment and Application of a LAMP Method for Visual Detection of Norovirus GⅡ in Aquatic Products
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1.College of Food Sciences and Technology, Shanghai Ocean University, Shanghai 201306, China;2.Technical Center for Animal, Plant and Food Inspection and Quarantine of Shanghai Customs District, Shanghai 200135, China;3.Shanghai Engineering Research Center of Aquatic Product Processing & Preservation, Shanghai 201306, China;4.Laboratory of Quality & Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture and Rural Affairs, Shanghai 201306, China;5.Engineering Research Center of Food Thermal-Processing Technology, Shanghai Ocean University, Shanghai 201306, China

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    摘要:

    【目的】基于环介导等温扩增（loop-mediated isothermal amplification，LAMP）建立一种快速检测水产品中GⅡ型诺如病毒的方法。【方法】针对诺如病毒的保守区域设计了4条引物，并对反应条件和反应体系进行优化。【结果】该反应的最佳体系为：Bst DNA聚合酶0.32 U/μL、MgSO4 10 mmol/L、dNTP 1.2 mmol/L、BIP 1.6 μmol/L、FIP 1.6 μmol/L、F3为0.4 μmol/L、B3为0.4 μmol/L；最佳反应条件为：63 ℃下反应50 min，随后升温至85 ℃继续反应5 min。建立的LAMP法能够特异性识别GⅡ型诺如病毒，其灵敏度为1×10-7 ng/μL，优于传统PCR法。另外，对3种可视化染料进行了测试，结果表明SYBR Green Ⅰ、苯酚红和羟基萘酚蓝（hydroxynaphthol blue，HNB）均对阳性和阴性结果有明显的颜色对比，因此可作为可视化染料。【结论】建立了一种快速、特异且操作简便的GⅡ型诺如病毒检测方法，已成功用于水产品的检测。

    Abstract:

    [Objective] This study aims to develop a method for the rapid detection of norovirus GⅡ based on loop-mediated isothermal amplification (LAMP). [Method] Four primers were designed based on the conserved region of norovirus and the reaction system and conditions were optimized. [Result] The optimal reaction system was composed of 0.32 U/μL Bst DNA polymerase, 10 mmol/L MgSO4, 1.2 mmol/L dNTP, 1.6 μmol/L BIP, 1.6 μmol/L FIP, 0.4 μmol/L F3, and 0.4 μmol/L B3. The optimal reaction conditions were reaction at 63 ℃ for 50 min and then at 85 ℃ for 5 min. The established LAMP method specifically recognized norovirus GⅡ, with the sensitivity of 1×10-7 ng/μL, which was better than that of conventional PCR. In addition, three visualization dyes were screened. The results showed that SYBR Green I, phenol red, and hydroxynaphthol blue (HNB) demonstrated significant color contrast between positive and negative results, indicating that all the three dyes could be used for visualization. [Conclusion] A fast, specific, and simple method is successfully established for detecting norovirus GⅡ and applied to the detection of aquatic products.

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姚婉婷,李想,谢庆超,欧杰,潘迎捷,赵勇,刘海泉.水产品中GⅡ型诺如病毒可视化LAMP法的建立及应用[J].食品与生物技术学报,2024,43(12):137-145.

YAO Wanting, LI Xiang, XIE Qingchao, OU Jie, PAN Yingjie, ZHAO Yong, LIU Haiquan. Establishment and Application of a LAMP Method for Visual Detection of Norovirus GⅡ in Aquatic Products[J]. Journal of Food Science and Biotechnology,2024,43(12):137-145.

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  • 收稿日期:2024-05-27
  • 最后修改日期:2024-08-30
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  • 在线发布日期: 2025-04-11
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