Abstract: The optimal preservation method and preservation time for DNA from the simulated water samples of reared fish was studied. Water samples at the same volume were pretreated with 0.45 μm filter membranes inanucleic acid-free environment. Comparison of the obtained quality of eDNA, PCR concentrations of fish 12S rRNA gene, and the amplicon sequencing results based on the available amplicon sequence variant method by combination of different preservation method and preservation time were studied. The results showed that with six preservation methods (－20 ℃ freezing, －80 ℃ freezing, ethyl alcohol at room temperature, 75% ethanol at room temperature, Longmire buffer at room temperature, and TK buffer at room temperature), the TK buffer at room temperature was the best eDNA captured methods, followed by freezing (－20 ℃, －80 ℃). And the －20 ℃, －80 ℃, and TK buffer at room temperature had good stability under the same preservation time respectively, and could stably preserve the filter membrane within two weeks to maximize the genetic information of fish in water samples. This study provided technical reference for third-party gene detection laboratories for fish eDNA preservation strategies and facilitating the development of eDNA metabarcoding technology.

Key words: environmental DNA, preservation conditions, DNA extraction, amplicon sequencing, available amplicon sequence variant method