Field application of an improved protocol for environmental DNA extraction, purification, and measurement using Sterivex filter

Abstract Environmental DNA (eDNA) is increasingly popular as a useful non-invasive method to monitor and study biodiversity and community structure in freshwater and marine environments. To effectively extract eDNA from the filter surface is a fundamental factor determining the representativeness of...

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Autores principales:	Marty Kwok-Shing Wong, Mako Nakao, Susumu Hyodo
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2020
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/9493a32f3cbb4e75a630406df5da3097
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spelling	oai:doaj.org-article:9493a32f3cbb4e75a630406df5da30972021-12-02T15:11:50ZField application of an improved protocol for environmental DNA extraction, purification, and measurement using Sterivex filter10.1038/s41598-020-77304-72045-2322https://doaj.org/article/9493a32f3cbb4e75a630406df5da30972020-12-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-77304-7https://doaj.org/toc/2045-2322Abstract Environmental DNA (eDNA) is increasingly popular as a useful non-invasive method to monitor and study biodiversity and community structure in freshwater and marine environments. To effectively extract eDNA from the filter surface is a fundamental factor determining the representativeness of the samples. We improved the eDNA extraction efficiency of an established Sterivex method by 12- to 16-fold using a larger volume of lysis buffer mix coupled with backflushing the cartridges. The DNeasy extraction column could be overloaded when the environmental sample input is high, possibly due to a higher nonspecific binding present in environmental samples, thus resulting in a relatively lower quantity measured. Therefore, we included an internal control DNA in the extraction to monitor the extraction and purification efficiencies in field samples, which is crucial for quantification of original eDNA concentration. The use of Takara Probe qPCR Mix supplemented with protein-based additives improved the robustness of the real time PCR assay on inhibitor-rich environmental samples, but prior purification by Qiagen PowerClean Pro Cleanup kit could be essential for inhibitor-rich water samples, even though the recovery rate was unexpectedly low (average 33.0%). The improved extraction and quantification complement the qualitative analyses including metabarcoding and metagenomics in field application.Marty Kwok-Shing WongMako NakaoSusumu HyodoNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 10, Iss 1, Pp 1-13 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Marty Kwok-Shing Wong Mako Nakao Susumu Hyodo Field application of an improved protocol for environmental DNA extraction, purification, and measurement using Sterivex filter
description	Abstract Environmental DNA (eDNA) is increasingly popular as a useful non-invasive method to monitor and study biodiversity and community structure in freshwater and marine environments. To effectively extract eDNA from the filter surface is a fundamental factor determining the representativeness of the samples. We improved the eDNA extraction efficiency of an established Sterivex method by 12- to 16-fold using a larger volume of lysis buffer mix coupled with backflushing the cartridges. The DNeasy extraction column could be overloaded when the environmental sample input is high, possibly due to a higher nonspecific binding present in environmental samples, thus resulting in a relatively lower quantity measured. Therefore, we included an internal control DNA in the extraction to monitor the extraction and purification efficiencies in field samples, which is crucial for quantification of original eDNA concentration. The use of Takara Probe qPCR Mix supplemented with protein-based additives improved the robustness of the real time PCR assay on inhibitor-rich environmental samples, but prior purification by Qiagen PowerClean Pro Cleanup kit could be essential for inhibitor-rich water samples, even though the recovery rate was unexpectedly low (average 33.0%). The improved extraction and quantification complement the qualitative analyses including metabarcoding and metagenomics in field application.
format	article
author	Marty Kwok-Shing Wong Mako Nakao Susumu Hyodo
author_facet	Marty Kwok-Shing Wong Mako Nakao Susumu Hyodo
author_sort	Marty Kwok-Shing Wong
title	Field application of an improved protocol for environmental DNA extraction, purification, and measurement using Sterivex filter
title_short	Field application of an improved protocol for environmental DNA extraction, purification, and measurement using Sterivex filter
title_full	Field application of an improved protocol for environmental DNA extraction, purification, and measurement using Sterivex filter
title_fullStr	Field application of an improved protocol for environmental DNA extraction, purification, and measurement using Sterivex filter
title_full_unstemmed	Field application of an improved protocol for environmental DNA extraction, purification, and measurement using Sterivex filter
title_sort	field application of an improved protocol for environmental dna extraction, purification, and measurement using sterivex filter
publisher	Nature Portfolio
publishDate	2020
url	https://doaj.org/article/9493a32f3cbb4e75a630406df5da3097
work_keys_str_mv	AT martykwokshingwong fieldapplicationofanimprovedprotocolforenvironmentaldnaextractionpurificationandmeasurementusingsterivexfilter AT makonakao fieldapplicationofanimprovedprotocolforenvironmentaldnaextractionpurificationandmeasurementusingsterivexfilter AT susumuhyodo fieldapplicationofanimprovedprotocolforenvironmentaldnaextractionpurificationandmeasurementusingsterivexfilter
_version_	1718387645567991808

Field application of an improved protocol for environmental DNA extraction, purification, and measurement using Sterivex filter

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