水产品中荧光假单胞菌(Pseudomonas fluorescens)RT-LAMP可视化检测方法的建立及应用  

Establishment and application of RT-LAMP visual detection method for Pseudomonas fluorescens in aquatic products

作　　者：魏梦泽 韩姣姣[1] 芦晨阳[1] 周君[1] 陈炯[1] 曲凌云 樊景凤[3] 陈刚[4] 苏秀榕[1] WEI Meng-ze;HAN Jiao-jiao;LU Chen-yang;ZHOU Jun;CHEN Jiong;QU Ling-yun;FAN Jing-feng;CHEN Gang;SU Xiu-rong(School of Marine Science,Ningbo University,Ningbo 315211,China;First Institute of Oceanography,Ministry of Natural Resources,Qingdao 266061,China;National Marine Environmental Monitoring Center,Dalian 116023,China;Fudan University,Shanghai 200433,China)

机构地区：[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

出　　处：《海洋科学》2018年第11期91-98,共8页Marine Sciences

基　　金：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~~

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(60min)和最适的Mg^2+(2.5mmol/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5.4copies/反应,比PCR检测方法高200倍。该方法结合SYBR-GreenI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。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 Mg^2+(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.

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 

分 类 号：TS254[轻工技术与工程—水产品加工及贮藏工程]