| 摘要: |
| 针对海蜇早期生活史阶段幼体种类鉴定困难的问题，建立一种快速、灵敏、操作简便的环介导等温扩增（LAMP）快速检测方法.根据海蜇的线粒体细胞色素C氧化酶Ⅰ基因（COⅠ）编码序列，设计LAMP引物，建立了海蜇幼体LAMP快速检测方法，并对LAMP反应体系、反应温度和反应时间进行了优化.结果表明，LAMP方法对海蜇目的基因的最小检测限为0.1 ng DNA.该检测方法特异性较强，与黄渤海其他常见钵水母种类海月水母、白色霞水母、沙海蜇均无交叉反应.利用本研究建立的海蜇LAMP检测方法对不同海域采集的成体海蜇样品和海蜇螅状幼体进行检测，结果显示，成体海蜇和海蜇幼体均呈现阳性结果，表明海蜇LAMP检测方法可以对海蜇幼体进行快速有效的检测.海蜇LAMP检测方法简单快速、灵敏且特异性强，可使其运用于沿海渔业部门海蜇资源的调查工作. |
| 关键词: 海洋生物学 海蜇 环介导等温扩增 快速检测 螅状幼体 |
| DOI：10.3969/J.ISSN.2095-4972.2017.04.010 |
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| 基金项目:国家自然科学基金资助项目（41576152） |
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| Development a rapid assay to detect the edible jellyfish Rhopilema esculentumlarvae using loop-mediated isothermal amplification method |
| SUN Ting-ting,LIU Zhong-yuan,LIANG Li-kun,DONG Zhi-jun |
| (College of Life Science, Yantai University, Yantai 264005, China;Yantai Institute of Coastal Zone Research Chinese Academy of Sciences，Yantai 264003, China) |
| Abstract: |
| It is difficult to identify the scyphomedusae larvae by traditional morphological methods, due to the complex morphological characteristics in different stages of their life cycle. For early identifying and detecting of this species, a simple and effective method using the loop-mediated isothermal amplification method (LAMP) has been developed. LAMP primers ofRhopilema esculentum were designed based on the mitochondrial cytochrome c oxidase subunit Ⅰ (COⅠ) gene. The amplification conditions, including the betaine and dNTPs concentrations, the inner primer to outer primer concentration ratio, reaction time and temperature, were optimized. The specificity, sensitivity and repeatability of the method were also tested. Our results show that the primers had specificity and repeatability. Samples from different geographic populations of R. esculentum and polyps produced positive results, while the other non-target scyphomedusae species produced negative results. In addition, the LAMP assay was more sensitive than conventional PCR. The detection limit of LAMP method was 0.1 ng DNA. Therefore, the established LAMP assay is a sensitive, specific, fast, and easily performed method for detection of the edible jellyfishR. esculentum. |
| Key words: marine biology Rhopilema esculentum loop-mediated isothermal amplification method rapid detection polyps |
