哈维氏弧菌LAMP可视化检测方法的建立  

Development of a rapid visual loop-mediated isothermal amplification(LAMP)method for detection of Vibrio harveyi

作　　者：于劼豪 黄郁葱[1,3] 孙良娟 钟键 鲁义善 简纪常[1,3] 蔡双虎 YU Jiehao;HUANG Yucong;SUN Liangjun;ZHONG Jian;LU Yishan;JIAN Jichang;CAI Shuanghu(Fisheries College of Guangdong Ocean University,Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture,Zhanjiang Guangdong 524088,China;Technology Center of Zhanjiang Customs,Zhanjiang Guangdong 524000,China;Shenzhen Institute of Guangdong Ocean University,Guangdong Provincial Engineering Technology Research Center for Aquatic AnimalHealth Assessment,Shenzhen Guangdong 518120,China)

机构地区：[1]广东海洋大学水产学院,广东省水产动物病害防控与健康养殖重点实验室,广东湛江524088 [2]湛江海关技术中心,广东湛江524000 [3]广东海洋大学深圳研究院,广东省水生动物健康评估工程技术研究中心,广东深圳518120

出　　处：《海洋渔业》2025年第1期29-39,共11页Marine Fisheries

基　　金：国家自然科学基金创新区域联合基金(U20A2065);广东省南美白对虾现代种业产业园项目(K22219);海关总署2022年科技项目(2022HK110)。

摘　　要：为提高哈维氏弧菌(Vibrio harveyi)早期快速诊断水平,建立一种基于环介导等温扩增(LAMP)技术的可视化快速检测方法。利用表达环二聚体磷酸二酯的VieA基因序列在哈维氏弧菌中的保守性,将其作为靶基因设计引物,并初步建立LAMP反应体系,对LAMP反应体系中各反应物浓度、内外引物比例、反应时间和反应温度进行优化,并对优化后的LAMP反应的特异性和灵敏度进行检测;分别在优化后的LAMP反应体系中加入SYBR Green I、钙黄绿素-锰离子或羟基萘酚蓝作为指标剂对LAMP反应产物进行可视化判别,从而建立一种针对哈维氏弧菌的LAMP可视化检测方法。研究表明,该LAMP反应体系最佳反应温度60℃、反应时间60 min、Mg2+浓度1.2 mmol·L^(-1)、dNTPs浓度0.64 mmol·L^(-1)、甜菜碱浓度0.25 mmol·L^(-1)及内外引物比例16∶1;该反应体系能特异性检测到哈维氏弧菌,且灵敏度为3.4×10-6 ng·μL^(-1)。可视化指标剂钙黄绿素-锰离子的添加比例为12∶1或羟基萘酚蓝的添加浓度为390μmol·L^(-1)时,LAMP反应产物的可视化效果最好,临床应用表明,该LAMP可视化技术能准确并特异性地检测到珍珠龙胆石斑鱼(Epinephelus fuscoguttatus♀×E.lanceolatus♂)和凡纳滨对虾(Litopenaeus vannamei)组织中感染的哈维氏弧菌。研究提供了一种简单、快速和结果可视化的哈维氏弧菌LAMP检测方法,可以为水生动物哈维氏弧菌的现场检测和弧菌病的早期诊断提供技术支持。Vibrio harveyi is an important member of marine microbial community and a common pathogenic bacterium that causes vibriosis in marine aquaculture.The detection and identification methods of V.harveyi mainly include traditional isolation and biochemical tests,immunological technique and molecular biology detection assay.However,the procedure of traditional detection and identification technique takes long time,immunological techniques and immunochromatography need expensive equipment and the procedure takes time,and molecular biology technique requires professional operation.Loop-mediated isothermal amplification(LAMP)is a versatile technique for detection and identification of microorganisms.In order to rapidly detect V.harveyi in early stage of vibirosis outbreak caused by V.harveyi,we explored the establishment of a visualized rapid detection method based on LAMP technology.The LAMP primers were designed based on the cyclic diguanylate phosphodiesterase VieA gene of V.harveyi as the target,and reaction time and temperature,concentrations of dNTPs,magnesium ion and betaine,and ratio of internal/external primers were optimized.The specificity and sensitivity of the optimized LAMP method were detected.SYBR Green I,calcein-manganese ion or hydroxynaphthol blue were added to the LAMP reaction system for visual discrimination of detecting V.harveyi-postive or V.harveyi-negative.The optimal reaction conditions were determined by the optimization of the conditions as follows:the temperature was 60℃,the reaction time was 60 min,the magnesium ion concentration is 1.2 mmol·L^(-1),the dNTPs concentration was 0.64 mmol·L^(-1),the betaine concentration was 0.25 mmol·L^(-1),and the ratio of internal/external primers was 16∶1,the addition ratio of calceinmanganese ions was 12∶1,and the addition concentration of hydroxynaphthol blue was 390μmol·L^(-1).The results showed that the established LAMP detection method specifically detected V.harveyi with a sensitivity of 3.4×10^(-6)ng·μL^(-1)(equivalent to 2.1×103 CFU�

关 键 词：哈维氏弧菌 LAMP技术 可视化检测 临床应用 

分 类 号：S947.9[农业科学—水产养殖]