Dataset Information

An optimised eDNA protocol for detecting fish in lentic and lotic freshwaters using a small water volume.

ABSTRACT: Environmental DNA is increasingly being used for assessing the presence and relative abundance of fish in freshwater, but existing protocols typically rely on filtering large volumes of water which is not always practical. We compared the effects of water volume, filtration type and eDNA extraction procedures in the detection of fish in three freshwater bodies (pond, lake and river) using a short fragment of the 12s rRNA mtDNA gene. Quantification of eDNA capture efficiency after DNA extraction, as well as amplification efficiency, were evaluated by conventional PCR and quantitative PCR. No significant differences on eDNA capture yield were found among freshwater bodies, but increasing water volume had a positive effect on eDNA capture and amplification efficiency. Although highest eDNA capture rates were obtained using 2 L of filtered water, 100 mL syringe filtration in combination with ethanol- sodium acetate precipitation proved to be more practical and increased quantitative PCR amplification efficiency by 6.4%. Our results indicate that such method may be optimal to detect fish species effectively across both lotic and lentic freshwater environments.

SUBMITTER: Muha TP

PROVIDER: S-EPMC6636732 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

An optimised eDNA protocol for detecting fish in lentic and lotic freshwaters using a small water volume.

Muha Teja Petra TP Robinson Chloe Victoria CV Garcia de Leaniz Carlos C Consuegra Sofia S

PloS one 20190717 7

Environmental DNA is increasingly being used for assessing the presence and relative abundance of fish in freshwater, but existing protocols typically rely on filtering large volumes of water which is not always practical. We compared the effects of water volume, filtration type and eDNA extraction procedures in the detection of fish in three freshwater bodies (pond, lake and river) using a short fragment of the 12s rRNA mtDNA gene. Quantification of eDNA capture efficiency after DNA extraction, ...[more]

PMID: 31314760

Similar Datasets

Project description:Flow cytometric methods for detecting and quantifying reduced intracellular thiol content using fluorescein-5-maleimide (F5M) in viable eukaryotic cells date back to 1983 (Durand and Olive [1]). There has been little development in these methodologies since that time, a period that has witnessed huge technological advances, particularly with the emergence of digital multi-parameter flow cytometric systems. Concurrent advancement in our understanding of redox regulation within eukaryotic cellular systems has also followed, whereby it is now accepted that cysteine thiols partake in redox reactions, which regulate protein activity and function (Groitl and Jakob (2014), Won et al. (2012)). Moreover, we are at the dawn of a new era in redox biology whereby the importance of 'reductive stress' in eukaryotic cellular systems is gathering momentum (Wadley et al. (2018) [4]). It is therefore critical that methods be continually advanced to better understand these concepts in more detail at the cellular level. Flow cytometry is a powerful technique that may be used for this purpose. Henceforth we have rejuvenated these methods to address modern scientific questions. In this paper, essential detail is provided on: •The adaption of a protocol initially described by Durand and Olive [1] for use with modern digital flow cytometer configurations. Here we provide optimal conditions for labelling intracellular thiols with F5M for detection using digital flow cytometers. Our modifications avoid the use of methanol fixation thus preserving cell viability in single cell suspension cultures.•Demonstration that flow cytometry can detect the gain and loss of reduced intracellular thiols in cells exposed to physiological doses of hydrogen peroxide mediated by glucose oxidase (Hole et al. (2013) [5]).•Validation of F5M protein labelling by coupling method to confocal microscopy and downstream proteomics, thus permitting a powerful experimental platform for potential use with next generation flow cytometry e.g. CyTOF (Lin and Maecker (2018) [6]).

Dataset Information

An optimised eDNA protocol for detecting fish in lentic and lotic freshwaters using a small water volume.

Publications

An optimised eDNA protocol for detecting fish in lentic and lotic freshwaters using a small water volume.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets