海蜇幼体环介导等温扩增快速检测方法的建立和应用

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 of Rhopilema 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 jellyfish R. esculentum.