环介导等温扩增技术快速检测施罗氏弧菌(Vibrio shilonii)  

Detection of Vibrio shilonii by using Loop-mediated isothermal amplification(LAMP)

作　　者：杨艺滢 陈偿[1,2,4] 罗鹏[1,2] 丁雄祺 谢媚 YANG Yi-Ying;CHEN Chang;LUO Peng;DING Xiong-Qi;XIE Mei(Key Laboratory of Tropical Marine Bio-resources and Ecology,South China Sea Institute of Oceanology,Chinese Academy of Sciences,Guangzhou,Guangdong 510301,China;Guangdong Provincial Key Laboratory of Applied Marine Biology,South China Sea Institute of Oceanology,Chinese Academy of Sciences,Guangzhou,Guangdong 510301,China;Xisha/Nansha Ocean Oberservation and Research Station,South China Sea Institute of Oceanology,Chinese Academy of Sciences,Guangzhou,Guangdong 510301,China;University of Chinese Academy of Sciences,Beijing 100049,China)

机构地区：[1]中国科学院南海海洋研究所中国科学院热带海洋生物资源与生态重点实验室,广东广州510301 [2]中国科学院南海海洋研究所广东省应用海洋生物学重点实验室,广东广州510301 [3]中国科学院大学,北京100049 [4]中国科学院南海海洋研究所西沙南沙深海海洋环境观测研究站,广东广州510301

出　　处：《微生物学通报》2018年第9期1871-1880,共10页Microbiology China

基　　金：中国科学院战略性先导科技专项(A类)资助(XDA13020302)~~

摘　　要：【背景】近年来,珊瑚白化事件频有发生,面临着严重衰退。由气候变化引起的珊瑚病原菌快速增殖是导致珊瑚白化的主要因素之一。施罗氏弧菌是枇杷珊瑚的致病菌,能侵入珊瑚虫体内而使珊瑚白化死亡。【目的】优化并建立一种钙黄绿素显色法快速检测珊瑚致病菌施罗氏弧菌的环介导等温扩增(Loop-mediatedisothermalamplificaiton,LAMP)检测技术。【方法】以枇杷珊瑚致病菌施罗氏弧菌为研究对象,针对施罗氏弧菌的rpoD (RNA polymerase subunit D)基因设计6条特异性扩增引物,建立LAMP检测体系并检测其特异性和灵敏度,同时对LAMP法、常规PCR和荧光定量PCR3种检测方法进行比较分析。【结果】供检测的10个样品菌株中,施罗氏弧菌反应结果为阳性,呈亮绿色,其他9株包括阴性对照(灭菌水为模板)反应结果为阴性,呈浅橙黄色;同时,所建立的钙黄绿素-LAMP方法最低检测限度为3.641×10~3 cps/mL,具有与荧光定量PCR等同的灵敏度和准确性,是常规PCR最低检测限度的0.1%;此外,通过模拟野外海水样品检测发现,钙黄绿素-LAMP方法对海水样品中施罗氏弧菌的检测限度可达1.3×10~2 CFU/mL。【结论】建立的钙黄绿素-LAMP检测技术具有很好的特异性、灵敏度和准确性,其操作方法简单、方便,无需昂贵仪器,适用于野外现场珊瑚致病菌施罗氏弧菌的快速检测。[Background] Coral bleaching events occur frequently which causes the coral reef undergoing severe degeneration in recent years. Disease caused by pathogens is one of the reasons for coral bleaching. Vibrio shilonii was identified to be a pathogen that is able to infect the coral Oculina patagonica and cause its bleaching. However, its epidermology remains largely unknown due to the lack of a rapid diagnostic tool. [Objective] To establish a rapid and simple method for detection of V. shilonii using Loop-mediated isothermal amplification （LAMP） technique. [Methods] A set of 6 specific primers based on the target of rpoD sequence was designed for LAMP amplification. The sensitivity and specification of the primers were tested and conditions for LAMP amplification were optimized. The products of LAMP were visualized with fluorescent reagent of calcein and agarose gel electrophoresis respectively. The limitation of the method was compared with conventional PCR and Real-time PCR. [Results] The result showed that this method can specifically detect V. shilonii without showing cross-reaction with closely related vibrio species or other bacteria. The detection limit is visualized with calcein was 3.641× 103 cps/mL of the plasmids carrying rpoD, which was 1 000 times more sensitive than conventional PCR and similar to that of Real-time PCR method; Meanwhile, this method was shown to be able to directly detect the pathogen in the seawater with a minimum concentration of 1.3× 102 CFU/mL. [Conclusion] Overall, this study revealed that the method we developed has high specificity and sensitivity for diagnosis of V. shilonii. It is more sensitive and more convenient than the conventional PCR or real-time PCR. Therefore, it has the potential to be used for diagnosis in the field.

关 键 词：枇杷珊瑚 施罗氏弧菌 环介导等温扩增 

分 类 号：Q93[生物学—微生物学]