鱼类环境DNA保存条件的比较研究

Study on the environmental DNA preservation conditions of fishes

为探索环境水样中鱼类DNA的最佳保存方法和保存时间,选取饲养鱼的水样为研究对象,在无核酸污染的环境中,采用0.45μm孔径滤膜对相同体积的水样进行预处理,并对富集环境DNA(eDNA)的滤膜进行不同保存方法和不同保存时间捕获的eDNA质量、鱼类12S rRNA基因文库浓度、基于ASVs方法的鱼类12S rRNA扩增子测序结果的比较研究。研究结果显示:-20℃冷冻、-80℃冷冻、无水乙醇-常温、75%乙醇-常温、Longmire’s保存液-常温以及TK保存液-常温等6种保存方法捕获的eDNA效果最好的是TK保存液-常温,其次为冷冻保存(-20℃、-80℃)。-20℃冷冻、-80℃冷冻和TK保存液-常温等3种保存方法在同一保存时间下具有良好的稳定性,且在保存时间2周内的测试条件下,也能稳定保存滤膜,最大程度还原水样中鱼类的遗传信息。研究对eDNA样本最佳保存条件的探索,将为第三方基因检测实验室对鱼类eDNA保存策略提供技术基础,便于eDNA宏条形码技术的开展。

The optimal preservation method and preservation time for DNA from the simulated water samples of reared fish was stud-ied.Water samples at the same volume were pretreated with 0.45μm 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 availa-ble 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 labo-ratories for fish eDNA preservation strategies and facilitating the development of eDNA metabarcoding technology.