College of Marine Sciences, Shanghai Ocean University, Shanghai, 201306, China; South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, 510300, China.
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, 510300, China; National Agricultural Experimental Station for Fishery Resources and Environment Dapeng, Shenzhen, 518120, China.
Mar Environ Res. 2022 Apr;176:105588. doi: 10.1016/j.marenvres.2022.105588. Epub 2022 Feb 28.
The development of a standardized eDNA detection process is the primary step in improving the accuracy and efficiency of eDNA detection. In this study, primers and probes with high specificity were selected to identify the COI gene of Acanthopagrus latus. Through experiments on the influence of different water quantities, methods of water sample preservation and water bathing times on the result of eDNA detection, the accuracy of this method for extracted water samples was improved. Specifically, a water bathing time of 6 h provided an optimal eDNA concentration from the water sample. After 6 h, the concentration began to decrease, so 6 h was determined to be the best water bathing time for A. latus. Five water extraction volumes (250 mL, 500 mL, 1 L, 2 L, and 3 L) were tested, and there was a positive correlation between water extraction volume and the DNA concentration in the water sample. Different water sample preservation methods were also compared, and it was found that at ≤7 d, the concentration obtained with the cryopreservation method for different water extraction volumes was higher than that obtained with the ethanol preservation method. In this study, we established and optimized a technical procedure for eDNA-based detection of A. latus in aquatic environments. We hope to apply this method in field investigations and provide a reference for the study of eDNA in other fishes.
建立标准化的 eDNA 检测流程是提高 eDNA 检测准确性和效率的首要步骤。本研究中，选择了具有高特异性的引物和探针来鉴定斜带石斑鱼的 COI 基因。通过对不同水量、水样保存方法和水浴时间对 eDNA 检测结果影响的实验，提高了该方法对提取水样的准确性。具体来说，水浴 6 h 为提取水样提供了最佳的 eDNA 浓度。6 h 后，浓度开始下降，因此确定 6 h 为斜带石斑鱼最佳的水浴时间。测试了 5 种水样提取体积（250 mL、500 mL、1 L、2 L 和 3 L），发现水样提取体积与水样中 DNA 浓度之间存在正相关关系。还比较了不同的水样保存方法，发现≤7 d 时，不同水样提取体积的冷冻保存方法获得的浓度高于乙醇保存方法。本研究建立并优化了一种基于 eDNA 的水生环境中斜带石斑鱼检测技术程序。我们希望将该方法应用于野外调查，并为其他鱼类的 eDNA 研究提供参考。
