Environmental DNA (eDNA) analysis has recently been used for detection of aquatic macro-organisms; however, the analytical procedures used in previous studies have not been optimized for practical use. Here, we compared several methods for DNA enrichment and extraction from water samples to establish widely applicable techniques for eDNA analysis using common carp as the model species. First, several types of filters were compared to identify the optimal filter type. Second, the eDNA yield was compared after a variety of extraction and isolation steps, including a combination of phenol extraction, ethanol precipitation (phenol treatment), and ultrafiltration. Third, DNA fixation with ethanol was tested for the preservation of eDNA on filters. Ethanol precipitation yielded the largest number of eDNA copies, followed by filtering using a 0.2-μm polycarbonate filter and a 0.7-μm glass fiber filter. Phenol treatment resulted in collection of a higher number of eDNA copies than that collected using ultrafiltration. DNA fixation with 15 ml ethanol enabled eDNA preservation on the filters at ambient temperatures for at least 6 days. Finally, combinations of different filter types and DNA enrichment procedures were compared using field water samples. From these results, we propose that the appropriate selection method for eDNA analysis should be chosen based on context. For example, when a high concentration of the target DNA is expected, such as in an aquarium experiment, ethanol precipitation is advantageous. However, when the target DNA is rare, which is the case in most field studies, filtration followed by freezing or DNA fixation by ethanol and phenol treatment are recommended. The filter type should be decided prior to the survey based on the characteristics of the water of interest. Thus, eDNA analysis could be applied to various situations using adaptive combinations of these techniques.
DNA extraction Environmental DNA Fish Quantitative PCR
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The authors sincerely thank Dr. M. Kondoh, Dr. H. Yamanaka, and the students in the Maruyama laboratory at Ryukoku University for discussions and comments on this study. This study was conducted as a part of the MEXT GRENEei Ecohealth Project (Project Leader: Prof. Chiho Watanabe, the University of Tokyo, Japan). This study was partly funded by the “Environmental Change and Infection Diseases in Tropical Asia” project (R-04) of the Research Institute for Humanity and Nature led by KM and by grants from JSPS KAKENHI (grant numbers 24657020 to TM and AM, and 26440238 to TM).
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