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)

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-mediated isothermal amplificaiton，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PCR 3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3.641×103 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1.3×102 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。

关键词:枇杷珊瑚，施罗氏弧菌，环介导等温扩增

Abstract:

[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.

Key words:Oculina patagonica, Vibrio shilonii, Loop-mediated isothermal amplification (LAMP)