Study On Interspecific Genetic Variation And Loop-mediated Isothermal Amplification Method For Detection Of Scyphomedusae From Bohai Sea And Yellow Sea

In recent years, blooms of the harmful scyphomedusae, which were severe nuisance to local fisheries, social production, and ecological environment, has become a prominent ecological problem in coastal waters of Bohai Sea and Yellow Sea. It is difficult to distinguish the scyphomedusae larvae by traditional classification methods, due to the complex morphological characteristics of different stages of their life cycle. This also restricts people to study the mechanism of its outbreak. Molecular biology techniques are not affected by different life stages and species status in species identification, which can effectively solve the problem in species identification, especially larvae identification problem. This study selected the common jellyfish included Aurelia aurita, Rhopilema esculentum, Nemopilema nomurai, Cyanea nozakii as research object, and find specific conserved sequences for molecular identification of Rhopilema esculentum and Cyanea nozakii, through analyzing the population genetic variation, and each LAMP detection method was established according to their conserved sequence.The analysis of mitochondrial cytochrome c oxidase I gene(COΙ) sequences shows, COΙ gene has right mutation rate, the largest intraspecific genetic distance is 0.015, while the interspecific genetic distance is about 0.197~0.270, significantly greater than the intraspecific genetic distance. The COΙ gene can be used as a reliable target sequence for detecting Cyanea nozakii and Rhopilema esculentum.LAMP primers of Cyanea nozakii were designed based on its COI gene. Change reaction conditions to optimize each reaction system. The specificity, sensitivity and repeatability of the method were tested. Experimental results showed that the primers had excellent specificity and repeatability, all samples of Cyanea nozakii produced positive results, while the other non-target scyphomedusae and hydrozoan species produced negative results. The detection limit of LAMP method was 0.1 ng DNA, displayed a higher amplification efficiency than traditional PCR.LAMP primers of Rhopilema esculentum were designed based on its COI gene. Change reaction conditions to optimize each reaction system. The specificity, sensitivity and repeatability of the method were tested. In addition, the test of detecting scyphistoma in asexual generation was conducted by established LAMP method. Experimental results show that the primers had excellent specificity and repeatability, samples from different geographic populations of Rhopilema esculentum produced positive results, while the other non-target scyphomedusae and hydrozoan species produced negative results. The detection limit of LAMP method was 0.1 ng DNA, the same with Cyanea nozakii. All hydroid of Rhopilema esculentum could be detected by LAMP method, which indicated LAMP method could be applied to molecular identification of hydroid.This study established a LAMP method for detecting Cyanea nozakii and Rhopilema esculentum respectively, according to their mitochondrial cytochrome c oxidase I gene. The method can be used to detect both jellyfish at different life stages, with good specificity, sensitivity and repeatability. Therefore, it may provide early warning of harmful jellyfish Cyanea nozakii and forecasting the resource of edible jellyfish Rhopilema esculentum.