文章摘要
基于toxR基因的轮虫弧菌荧光定量微流控快速检测技术的建立
Establishment of a Rapid Fluorescence Quantitative Microfluidic Detection Technique for Vibrio rotiferianus Based on toxR Gene
投稿时间:2019-12-01  修订日期:2020-01-02
DOI:
中文关键词: 轮虫弧菌  toxR  实时荧光定量PCR  微流控快速检测
英文关键词: Vibrio rotiferianus  toxR  quantitative real-time PCR  Microfluidic rapid detection
基金项目:国家重点基础研究规划项目
作者单位E-mail
陈京 上海海洋大学 水产与生命学院 上海 79881763@qq.com 
于永翔 中国水产科学研究院黄海水产研究所  
张正 中国水产科学研究院黄海水产研究所 zhangzheng@ysfri.ac.cn 
王印庚 中国水产科学研究院黄海水产研究所  
廖梅杰 中国水产科学研究院黄海水产研究所  
荣小军 中国水产科学研究院黄海水产研究所  
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中文摘要:
      本文以轮虫弧菌的单拷贝基因toxR基因的高保守区域为目的片段,设计特异性引物,构建重组质粒标准品,建立针对该菌的荧光定量PCR检测方法。以此为基础,选用UF-150 Genechecker微流控荧光定量PCR仪,通过优化反应体系和反应条件,建立了轮虫弧菌的微流控荧光定量PCR检测方法。该方法能特异性扩增toxR基因目的片段,对轮虫弧菌纯培养物检测下限为1.34×100 copies/μL。在人工感染样品中的应用结果表明:对鱼体组织中轮虫弧菌的检测下限为1.34×103 cfu/mL,检测结果可以目视判读,检测时间缩短至42 min以内。该检测方法具有特异性强、敏感度高、场地要求低等突出优势,适于开发水产病原实用性的现场快速检测技术。
英文摘要:
      The highly conserved region of Vibrio rotiferianus toxR gene was selected as the target fragment for the specific primers design. The recombinant plasmid was constructed based on the primers and established a fluorescence quantitative PCR method for V. rotiferianus . And UF-150 Genechecker microfluidic quantitative real-time PCR instrument was used as a platform to develop a microfluidic quantitative real-time PCR detection method for V. rotiferianus with the optimize reaction system and reaction conditions. The results indicated that, this method can specifically amplify toxR gene target fragment, and the minimum detection for V. rotiferianus is 1.34×100 copies/μL. Artificial infection detection results show that the minimum detected contribution for V. rotiferianus in fish tissues is 1.34×103 cfu/g. The results can be visually interpreted and the detection time was less than 42 min. This method has prominent advantages of strong specificity, high sensitivity and low site requirements, it is suitable for developing practical field rapid detection technology of aquatic pathogens.
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