魏梦泽¹, 韩姣姣¹, 芦晨阳¹, 周君¹, 陈炯¹, 曲凌云², 樊景凤³, 陈刚⁴, 苏秀榕¹
1.宁波大学海洋学院, 浙江 宁波 315211;2.自然资源部第一海洋研究所, 山东 青岛 266061;3.国家海洋环境监测中心, 辽宁 大连 116023;4.复旦大学, 上海 200433

摘要:
荧光假单胞菌（Pseudomonas fluorescens）在低温储存的水产品中仍能存活并繁殖，是导致水产品品质下降和恶化的主要原因。因此开发一种快速高效的检测手段显得尤为迫切。本文将逆转录-环介导等温扩增（Reverse Transcription Loop-mediated Isothermal Amplification，RT-LAMP）技术与可视化检测手段结合，通过条件优化，确定了最佳反应温度（63℃），反应时间（60 min）和最适的Mg²⁺（2.5 mmol/L）添加量，建立了快速、高效、灵敏的RT-LAMP检测方法。灵敏度检测表明：本方法最低检出限为5.4 copies/反应，比PCR检测方法高200倍。该方法结合SYBR-Green I染色，通过目测就可以直接鉴别荧光假单胞菌，使得检测结果更加直观，简便可行。
关键词:荧光假单胞菌逆转录-环介导等温扩增活菌检测可视化检测
DOI：10.11759/hykx20180221001
分类号:TS254
基金项目:海洋公益性行业专项经费资助项目（201105007）；海洋经济创新发展区域示范项目

Establishment and application of RT-LAMP visual detection method for Pseudomonas fluorescens in aquatic products
WEI Meng-ze¹, HAN Jiao-jiao¹, LU Chen-yang¹, ZHOU Jun¹, CHEN Jiong¹, QU Ling-yun², FAN Jing-feng³, CHEN Gang⁴, SU Xiu-rong¹
1.School of Marine Science, Ningbo University, Ningbo 315211, China;2.First Institute of Oceanography, Ministry of Natural Resources, Qingdao 266061, China;3.National Marine Environmental Monitoring Center, Dalian 116023, China;4.Fudan University, Shanghai 200433, China
Abstract:
Live Pseudomonas fluorescens is the main reason for the deterioration of aquatic products even in low-temperature storage. Therefore, there is an urgent need to develop a rapid and efficient detection method. In this study, we combined reverse transcription loop-mediated isothermal amplification (RT-LAMP) technology with visual detection methods, and determined the optimum reaction temperature (63℃), reaction time (60 min), and the optimum addition of Mg²⁺ (2.5 mmol/L) by optimizing the conditions. Thus, a rapid, efficient, and sensitive RT-LAMP detection method was established. A sensitivity test showed that RT-LAMP could be identified as low as 5.4 copies per reaction, which is about 200-fold more sensitive than the standard polymerase-chain-reaction detection method. This method can be used to effectively identify P. fluorescens and the RT-LAMP results can be visually inspected using SYBR Green I staining in a closed-tube, which makes the detection results intuitive and simple to understand.
Key words:Pseudomonas fluorescensRT-LAMPdetection of live bacteriavisualized detection





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