大菱鲆弧菌LAMP快速检测方法的建立与初步应用

The Development of a Loop-mediated Isothermal Amplification(LAMP)Method for Detection of Vibrio scophthalmi and its Initial Application

为建立大菱鲆弧菌环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测方法,本试验以大菱鲆弧菌的luxR基因为靶点设计LAMP扩增引物,通过对反应时间和反应温度的优化,成功建立了大菱鲆弧菌LAMP检测方法,并研究了该方法的特异性、灵敏度和应用性。结果显示:应用该LAMP方法在62℃反应30min,可成功检测到大菱鲆弧菌的存在。选择6种细菌菌株作为检测模板,发现除大菱鲆弧菌反应结果呈阳性外,其它均为阴性,说明该检测方法特异性良好。灵敏度检测结果显示该方法比普通PCR检测方法灵敏度高102倍。以大菱鲆作为实验动物,检测该LAMP技术的实际应用性,结果显示该方法可准确检测并鉴定出组织和血液中感染的大菱鲆弧菌,其灵敏度可达7~14CFU/反应。在反应产物中加入荧光染料后,检测结果极易用肉眼判别。本研究成功建立了大菱鲆弧菌LAMP检测方法,该方法可用于水产养殖中大菱鲆弧菌的早期诊断。

To establish a loop-mediated isothermal amplification(LAMP)detection method for Vibrio scophthalmi,four specific primers were designed based on DNA sequence of V.scophthalmi uxR gene.The LAMP detection method was established successfully after optimization of reaction temperature and time.After that,experiments were carried out to study its specificity,sensitivity and application.Research findings showed that the optimized reaction temperature and time was 62℃and 30 min,respectively.The sensitivity of this method is 102 times higher than that of general PCR method.The specificity of this method was validated against a total of 6vibrio strains,in which only V.scophthalmi could be detected successfully.The results of application study showed that using this method,V.scophthalmi infected into turbot tissues and blood could be detected accurately,and its sensitivity was 7~14CFU/reaction.Moreover,the LAMP results were visualized after fluorescent dye was added into the tubes.These results indicated that the LAMP detection method of V.scophthalmi was successfully established,and it can be used for the early diagnosis of pathogenic bacteria in aquaculture.