Isopropanol precipitation method for collecting fish environmental DNA

Abstract

The environmental DNA (eDNA) method has been used to estimate the distributions of aquatic species, and both ethanol precipitation and filtration‐based methods are commonly employed to capture eDNA from sampled water. Although filtration‐based methods can capture more eDNA than that by ethanol precipitation, by processing larger volumes of water (e.g., 1 L vs. 15 mL), ethanol precipitation can immediately preserve eDNA on site and ease downstream processing, which are especially advantageous for eDNA studies conducted with limited resources, such as electric equipment, labor, and time. However, the ethanol precipitation method is limited by small volume of water that can be processed (i.e., 15 mL in a reaction volume of 50 mL). As an alternative, isopropanol could potentially be used to increase the volume of sample water processed, since lower volumes of isopropanol are required for precipitation. Therefore, in the present study, we compared the copy numbers of carp eDNA captured using isopropanol and ethanol precipitation in both mesocosm and field experiments. In both cases, we found that isopropanol precipitation recovered double the amount of eDNA recovered by ethanol precipitation, when the reaction volumes were equal. Therefore, isopropanol precipitation is a superior option for eDNA capture when surveys are conducted under resource‐limited conditions.

Standards for Methods Utilizing Environmental DNA for Detection of Fish Species

“…The methods of eDNA concentration involve centrifugation, isopropanol or ethanol precipitation, and filtration. Centrifugation and precipitation are generally suitable for collecting small volumes such as 1.5 mL [34] or 15 mL [48] of water, whereas filtration can handle larger bulk volumes from 250 mL to 45 L of water (Table S2). Filtration of a large volume of water can improve the detection of rare species, and preservation of the filters is more convenient than using water samples; hence, filtration methods have become the most widely used methods for fish eDNA capture from water samples.…”

“…In fish eDNA detection, samples are collected from diverse water sources including artificial or experimental sources, ponds, lakes, streams, rivers, and seas and are obtained from different depths including the surface, middle, bottom, or others (Table S2). Sample volumes vary from 1.5 mL [34] to 45 L [35]. However, 1 or 2 L is typically collected from the field (Table S2).…”

The detection of aquatic macroorganisms using environmental DNA analysis—A review of methods for collection, extraction, and detection

“…Thus, ethanol precipitation would be suitable when the eDNA concentration of the target species is high (Doi, Uchii, et al, ). As an alternative, isopropanol precipitation can be used for eDNA collection (Doi et al, ). This approach increases the processable volume of sample water in the reaction volume, since a lower volume of isopropanol is required for precipitation compared with ethanol.…”

The effect of filtration method on the efficiency of environmental DNA capture and quantification via metabarcoding

“…Capturing eDNA through precipitation entails adding ethanol or isopropanol with sodium acetate to water samples (Dejean et al., ; Doi et al., ; Foote et al., ). Samples can be preserved quickly and easily in the field using such an approach, but it is only feasible for small volumes of water (<30 ml), which could reduce the probability of detection, particularly of rare species (Deiner, Walser, Machler, & Altermatt, ; Eichmiller, Miller, & Sorensen, ).…”