食品科学 ›› 2020, Vol. 41 ›› Issue (10): 288-296.doi: 10.7506/spkx1002-6630-20190125-323

• 安全检测 • 上一篇    下一篇

副溶血性弧菌毒素编码基因的微量、可视化检测

苏晨丽,陈兰明   

  1. (上海海洋大学食品学院,上海 201306)
  • 出版日期:2020-05-25 发布日期:2020-05-15
  • 基金资助:
    国家自然科学基金面上项目(31671946);上海市科委地方能力建设项目(17050502200)

Visual and Trace Detection of Toxin Genes of Vibrio parahaemolyticus

SU Chenli, CHEN Lanming   

  1. (College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China)
  • Online:2020-05-25 Published:2020-05-15

摘要: 针对副溶血性弧菌主要毒素编码基因(tdh和trh),基于环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术,以毛细管为反应介质,建立一种微量(5 μL)、特异、灵敏、低成本、可视化、快速检测的毛细管LAMP(capillary LAMP,cLAMP)。本研究针对每个靶标基因分别设计6 条特异性引物,系统优化Mg2+、dNTPs浓度和Bst DNA聚合酶使用量,以及反应时间及反应温度等参数,确立最适cLAMP反应体系;测定该方法的灵敏度和特异性。结果表明:优化后的5 μL cLAMP反应体系为6 mmol/L或8 mmol/L Mg2+,1.44 mmol/L或1.28 mmol/L dNTPs、0.096 U/μL Bst DNA聚合酶、反应温度65 ℃、反应时间60 min。针对靶基因tdh和trh,cLAMP方法检测副溶血性弧菌基因组DNA的最低检测限分别为3 fg/μL和34 fg/μL,纯培养物的最低检测限分别为9.85×103 CFU/mL和8.25×105 CFU/mL;且与其他6 种常见致病菌(霍乱弧菌、创伤弧菌、溶藻弧菌、嗜水气单胞菌、大肠杆菌、金黄色葡萄球菌)均无交叉反应。本研究建立的微量、可视化cLAMP方法为食源性致病菌副溶血性弧菌检测试剂盒的研发提供了技术支撑。

关键词: 副溶血性弧菌, 毒素编码基因, 毛细管环介导等温扩增, 可视化检测, 微量检测

Abstract: Based on loop-mediated isothermal amplification (LAMP), a sensitive, specific, low-cost, visual and trace method was developed using a capillary as the reaction medium for the detection of the toxin genes tdh and trh of Vibrio parahaemolyticus. Six primer pairs were designed targeting either of the toxin genes. The concentrations of Mg2+, dNTPs and Bst DNA polymerase, reaction time and temperature were optimized for the capillary LAMP (cLAMP) method. The sensitivity and specificity of the method were evaluated. The results indicated that optimum conditions of the cLAMP method were as follows: magnesium ion concentration 6 or 8 mmol/L dNTPs cocnetration 1.44 or 1.28 mmol/L, Bst DNA polymerase concentration 0.096 U/L (in a volume of 5 μL), reaction temperature, 65 ℃, and time, 60 min. The limits of detection (LODs) of the tdh and trh genes were 3 and 34 fg/μL for genomic DNA, and 9.85 × 103 and 8.25 × 105 CFU/mL for bacterial culture, respectively. No cross-reactivity was observed with other common bacterial pathogens, including cholera, V. vulnificus, V. alginolyticus, Aeromonas hydrophila, Escherichia coli, and Staphylococcus aureus. The results in this study provide technical support for the development of visual and trace detection kits for the foodborne pathogen V. parahaemolyticus.

Key words: Vibrio parahaemolyticus, toxin-encoding genes, capillary loop-mediated isothermal amplification, visual detection, trace detection

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