Capture of Environmental DNA (eDNA) from Water Samples by Flocculation

Summary

Environmental DNA (eDNA) is typically captured from water samples using alcohol precipitation, filtration, or flocculation. Presented here is an alternative protocol that uses lanthanum chloride to enable collection of dissolved and particulate-bound nucleic acids from water samples containing eukaryotic cells, prokaryotic cells, and virus.

Abstract

The analysis of environmental DNA (eDNA) has become a widely used approach to problem solving in species management. The detection of cryptic species including invasive and (or) species at risk is the goal, typically accomplished by testing water and sediment for the presence of characteristic DNA signatures. Reliable and efficient procedures for the capture of eDNA are required, especially those that can be performed easily in the field by personnel with limited training and citizen scientists. The capture of eDNA using membrane filtration is widely used currently. This approach has inherent issues that include the choice of filter material and porosity, filter fouling, and time required on site for the process to be performed. Flocculation offers an alternative that can be easily implemented and applied to sampling regimes that strive to cover broad territories in limited time.

Introduction

Before the mid-2000s, the term “environmental DNA” was generally used to refer to DNA obtained from water and soil microbes although some use for the detection of human and animal DNA for purposes of fecal source tracking or non-native species detection had begun^1,2,3. Presently, environmental DNA is generally used to describe DNA from cells sloughed from eukaryotic organisms, and the analysis of this DNA has become a widely used management tool. The detection of cryptic species including those that are invasive or species at risk is the goal, typically accomplish....

Protocol

1. Decontamination of equipment and flocculation reagent preparation

1. Thoroughly wash all bottles, tubing, and other previously used equipment with dishwashing detergent and hot water.
2. Spray all bottles, tubing, and other previously used equipment with 6% citric acid solution to remove mineral deposits, let stand 20 minutes, and rinse with tap water.
3. Add a solution of 10-fold diluted household bleach (0.5-0.6% final concentration of sodium hypochlorite) to all bottles (approximately 10% of the container volume), shake, let it stand at least 1 h, then drain and rinse with tap water until no trace of bleach remains. Then rinse once with de....

Results

Eight 500 mL water samples (450 mL each) taken from an aquarium housing two Eastern painted turtles (Chrysemys picta picta) were used to prepare eDNA using the described protocol. Another eight 500 mL water samples were filtered over 47 mm glass fiber filters of 1.5 µm porosity containing no binder and eDNA was extracted using the same extraction method as used for the flocculation protocol. DNA prepared using both methods was analyzed using quantitative PCR that targets the mitochondrial control region of the t.......

Discussion

Although this technique is straightforward, some steps are critical to success. The collection and transfer of the flocs must be done completely so as not to lose DNA. Residual material must be gathered using strategic rinses. The described protocol uses a commercial DNA extraction kit for the final processing of flocculated material. Other extraction methods may perform similarly or may be superior in performance, but modifications of this part of the protocol have not been exhaustively tested.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5M EDTA, pH 8.0	Available from several commercial sources
1 x TE Buffer, pH 8.0	Available from several commercial sources
15 ml Conical Centrifuge Tubes	Single use
16 oz. PET Clear Square Bottles	Reusable with cleaning and decontamination
38/400 Polypropylene Cap with Pressure Sensitive Liner	Leave caps loosely attached until use. Cap tightly to seal pressure sensitive liner.
50 mL Conical Centrifuge Tubes	Single use
6% Citric Acid Solution	Use to remove mineral deposits from reusable sample bottles.
Fecal/Soil Microbe Kit	Use inhibitor removal resin or column
Lanthanum (III) chloride heptahydrate	100 mM Stock Solution
Peristaltic DC Pump	Any peristaltic pump will do, or can be syphoned
Polyacryl Carrier	10% linear polyacrylamide
Sodium Bicarbonate	1 M, filter sterilized and stored at room temperaruture