创伤弧菌和副溶血弧菌双重LAMP检测方法的建立及初步应用  

The development and application of a duplex loop-mediated isothermal amplification method for rapid detection of Vibrio vulnificus and Vibrio parahaemolyticus

作　　者：周顺[1,2] 高志鑫[2] 张敏[2] 

机构地区：[1]中国海洋大学水产学院,山东青岛266100 [2]青岛农业大学海洋科学与工程学院,山东青岛266109

出　　处：《中国兽医学报》2016年第11期1875-1881,共7页Chinese Journal of Veterinary Science

基　　金：山东省农业重大应用技术创新项目;山东省现代农业产业技术体系鱼类产业创新团队项目

摘　　要：为建立创伤弧菌和副溶血弧菌的双重环介导等温扩增（loop—mediated isothermal amplification，LAMP）检测方法，本试验以创伤弧菌metalloprotease基因和副溶血弧菌ompA基因为靶点设计LAMP扩增引物，并在内引物间添加不同的酶切位点，通过对反应时间和温度的优化及酶切反应，成功建立了双重LAMP检测方法，并对其检测特异性、灵敏度和实际应用性进行了研究。结果显示，62℃反应45min时可同时检测到2种弧菌的存在，通过限制性内切酶酶切，可准确区分2种弧菌。该方法比普通PCR检测方法灵敏度高10^2～10^3倍。选择17株细菌菌株作为检测模板，发现除创伤弧菌和副溶血弧菌反应结果呈阳性外，其他均为阴性。以大菱鲆作为实验动物，检测双重LAMP技术的实际应用可能，该方法可准确检测并鉴定出组织和血液中感染的细菌种类，其灵敏度可达374～410CFU／g（mL）。SYBR Green Ⅰ是一种结合于DNA双链小沟中的荧光染料，反应产物加入该染料后，极易用肉眼判别。本试验成功建立了创伤弧菌和副溶血弧菌的双重LAMP检测方法，该方法可用于水产养殖中病原菌的早期诊断。In this study, two sets of primers were designed to construct a duplex loop-mediated isothermal amplification （LAMP） method for simultaneously detection of Vibrio vulnificus and Vibrio parahaemolyticus. The results showed that when the mixture were incubated for 45 rain at 62℃ ,the duplex method can simultaneously detect the presence of two Vibrios, which can be dis- criminated by subsequent restriction analysis. The sensitivity of the duplex LAMP method was 10^2-10^3 folds higher than that of PCR method. The specificity of this method was validated in a total of 17 bacterial strains,in which only V. vulnificus and V. parahaernolyticus can be positively detected. In order to investigate the application potential of this method, turbot was used as trial animal,and healthy and infected tissues were detected with the duplex LAMP method. The results showed the detection sensitivity of the method was 374-410 CFU/g （mL）. Moreover,the reaction products were visualized after fluorescent dye was added. Above results indicated that a duplex LAMP method for simultaneously detection of V. vulnificus and V. Parahaemolyticus was successfully constructed, which can be used for the early diagnosis of pathogenic bacteria in aquaculture.

关 键 词：创伤弧菌 metalloprotease基因 副溶血弧菌 ompA基因 双重LAMP 

分 类 号：S852.61[农业科学—基础兽医学]