Name: use of a filter cartridge for filtration of water samples and extraction of environmental dna
Uploaded: 2016-11-25T13:00:00
Description: Watch this Scientific Journal Video about Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA at JoVE.com

This study was supported as basic research by CREST from the Japan Science and Technology Agency (JST) and by grants from JSPS/MEXT KAKENHI (Number 26291083) and the Canon Foundation to M.M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

NOTE: Ce protocole ne traite pas des méthodes d&#39;échantillonnage de l&#39;eau et metabarcoding. L&#39; eau peut être échantillonné de différentes manières en fonction des fins d&#39;étude 16 et voir Miya et al. 12 pour plus de détails sur les méthodes de metabarcoding en utilisant des amorces de MiFish. A noter que l&#39;eau prélevée doit rester très froid et on le filtre en quelques heures pour éviter une dégradation de EdNA. A noter également que ce protocole implique ...

<ol>
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Dans de nombreuses études de metabarcoding en utilisant des échantillons environnementaux tels que l&#39; eau et le sol, le traitement post-filtration de la cartouche filtrante est généralement la suivante 24,25: 1) de coupe ouverte ou gerçures le boîtier avec des outils à main (coupe-tube ou une pince); 2) le retrait du filtre de la cartouche; et 3) la découpe du filtre en petits morceaux avec une lame de rasoir pour l&#39;extraction d&#39;ADN. Pour éviter de telles lourdes et longues procédures qu...

ADN de l&#39;environnement (EDNA) dans les milieux aquatiques se réfère au matériel génétique trouvé dans la colonne d&#39;eau. Des études récentes ont démontré l&#39;utilité de EDNA pour détecter les poissons de divers milieux aquatiques, y compris les étangs, les rivières 1-3 4-8, cours d&#39; eau 9, et l&#39; eau de mer 10-14. La plupart de ces études se sont concentrées sur la détection d&#39;un seul ou de quelques invasive 1,4-6,8,14 et rares ou menacées espèces 3,9, alors que certaines études récentes ont tenté la détection simultanée de plusieurs espèces dans les communautés de poissons locales 7,9, 12,13,15 et mésocosmes 11,12. Cette dernière approche est appelée «metabarcoding» et EdNA metabarcoding utilise un ou plusieurs ensembles d&#39;amorces de PCR pour coamplify une région de gène à travers taxonomiquement divers échantillons. Elle est suivie par la préparation bibliothèque d&#39;indexation et l&#39;addition de l&#39;adaptateur et les bibliothèques indexées sont analysées par un séquençage parallèle à haut débitPlate-forme. Récemment Miya et al. 12 ont développé des amorces de PCR universelles pour metabarcoding EDNA de poissons (appelés &quot;MiFish&quot;). Les amorces MiFish ciblent une région hypervariable du gène 12S ARNr mitochondrial (163-185 pb), qui contient des informations suffisantes pour identifier les poissons à la famille taxonomique, genre et espèce, sauf pour certains congénères étroitement liés. Avec l&#39;utilisation de ces amorces dans EDNA metabarcoding, Miya et al. 12 détecté plus de 230 espèces marines subtropicales de réservoirs d&#39;aquarium avec la composition des espèces et des récifs coralliens connus près de l&#39;aquarium. Tout en optimisant le protocole metabarcoding pour accueillir l&#39;eau de mer naturelle avec des niveaux de concentration de poissons EDNA différents, nous avons remarqué que les amorces MiFish parfois échoué à amplifier la région cible pour la préparation de la bibliothèque ultérieure. Une des raisons les plus probables pour cette amplification PCR échec est le manque de quantités suffisantes de la template ADN contenu dans de petits volumes d&#39;eau filtrée (ie 1-2 L). Bien que la concentration EDNA d&#39;un groupe taxonomique spécifique est inconnaissable avant l&#39;amplification, la filtration de grands volumes d&#39;eau (&gt; 1-2 L) serait un moyen simple et efficace pour recueillir plus EDNA des milieux aquatiques avec l&#39;abondance des poissons rares et la biomasse, tels que ouvert sur la mer et en eaux profondes des écosystèmes. Par rapport aux filtres en fibre de disques utilisés classiquement dans un certain nombre de recherches poissons EDNA 16, les cartouches filtrantes ont l&#39;avantage d&#39;accueillir de plus grands volumes d&#39;eau avant le colmatage 17. En fait, une étude récente a montré un grand volume (&gt; 20 L) filtration des échantillons d&#39;eau de mer côtières en utilisant des cartouches filtrantes 18. En outre, ils sont emballés individuellement et stérile, ainsi que plusieurs étapes du flux de travaux expérimentaux peuvent être réalisés dans le boîtier de filtre, ce qui réduit la probabilité de contamination du laboratoire 19. Le dernierfonctionnalité est essentielle pour EDNA metabarcoding, où le risque de contamination reste parmi les plus grands défis expérimentaux 20,21. En dépit de ces avantages techniques des cartouches filtrantes, il n&#39;a pas été utilisé dans les études EDNA de poissons à deux exceptions près 8,15. Ici, nous fournissons un protocole pour la filtration des échantillons d&#39;eau avec la cartouche filtrante et l&#39;extraction de EDNA de son filtre sans avoir à couper ouvrir le boîtier. Nous fournissons également deux systèmes de filtration d&#39;eau de remplacement en fonction des volumes d&#39;eau (≤4 L ou&gt; 4 L). Pour comparer les performances du protocole nouvellement développé et un protocole précédemment utilisé à l&#39; aide d&#39; un filtre en fibre de verre dans notre groupe de recherche 12,14,22,23, nous effectuons EDNA metabarcoding analyse de l&#39; eau de mer à partir d&#39; un énorme réservoir d&#39;aquarium (7.500 m 3 ) avec la composition des espèces connues, et indiquer le nombre d&#39;espèces détectées provenant des deux protocoles que des résultats représentatifs. Ce protocole hcomme été développé pour metabarcoding EDNA de poissons, mais est également applicable à EDNA d&#39;autres organismes.

<table border="0" cellpadding="0" cellspacing="0" width="927">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Mesh panel</td>
			<td style="width:167px;">Iris Ohyama</td>
			<td style="width:119px;">MPP-3060-BE</td>
			<td style="width:411px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Metal prong</td>
			<td style="width:167px;">Iris Ohyama</td>
			<td style="width:119px;">MR12F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Stand for the mesh panel</td>
			<td style="width:167px;">No brand</td>
			<td>4184-9507</td>
			<td>available from Amazon Japan</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">1-L plastic bag with screw cap</td>
			<td style="width:167px;">Yanagi</td>
			<td style="width:119px;">DP16-TN1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Male luer-lock connector</td>
			<td style="width:167px;">ISIS</td>
			<td style="width:119px;">11620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">10-mL pipette tip</td>
			<td style="width:167px;">Eppendorf</td>
			<td style="width:119px;">0030 000.765</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">10-L book bottle with valve</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">1-2169-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Sterivex-HV filter</td>
			<td style="width:167px;">Millipore</td>
			<td style="width:119px;">SVHVL10RC</td>
			<td>denoted as &#34;filter cartridge&#34; throughout the ms and used in the protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Male luer fitting</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">1-7379-04</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Female luer fitting</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">5-1043-14&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Inlet luer cap</td>
			<td style="width:167px;">ISIS</td>
			<td style="width:119px;">VRMP6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Outlet luer cap</td>
			<td style="width:167px;">ISIS</td>
			<td style="width:119px;">VRFP6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">High vacuum tubing</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">6-590-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Vacuum connector</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">6-663-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone stopper</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">1-7650-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Manifold</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">2-258-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Aspirator-GAS-1</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">1-7483-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">DNeasy Blood &#38; Tissue Kit (250)</td>
			<td style="width:167px;">Qiagen</td>
			<td style="width:119px;">69506</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">PowerWater Sterivex DNA Isolation Kit</td>
			<td style="width:167px;">MO BIO</td>
			<td style="width:119px;">14600-50-NF</td>
			<td>denoted as &#34;optional kit&#34; in the ms</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Tabletop Centrifuge</td>
			<td style="width:167px;">Kubota</td>
			<td style="width:119px;">Model 4000</td>
			<td>Maximum speed 6,000 rpm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Fixed-angle rotor</td>
			<td style="width:167px;">Kubota</td>
			<td style="width:119px;">AT-508C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Adaptor for a 15 mL conical tube</td>
			<td style="width:167px;">Kubota</td>
			<td style="width:119px;">055-1280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">RNAlater Stabilization Solution</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM7020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;"></td>
			<td style="width:167px;">Parafilm</td>
			<td style="width:119px;">PM992</td>
			<td>denoted as &#34;self-sealing film&#34;</td>
		</tr>
	</tbody>
</table>

use of a filter cartridge for filtration of water samples and extraction of environmental dna

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

Il est techniquement difficile d&#39;isoler et de quantifier seulement EDNA de poissons de la EDNA en vrac extrait, parce que les amorces MiFish coamplify la région cible de certains vertébrés non-poissons, tels que les oiseaux et les mammifères, avec des produits de PCR de la même taille (env. 170 pb ) 12. Au lieu de quantifier les poissons EDNA, nous effectuons MiFish metabarcoding analyse de EDNA à partir d&#39;un réservoir d&#39;aquarium avec la composition...

Watch this Scientific Journal Video about Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA at JoVE.com

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA (Video) | JoVE

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

Utilisation d&#39;une cartouche filtrante pour la filtration de l&#39;eau et des échantillons d&#39;extraction de l&#39;ADN de l&#39;environnement

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

Research

JoVE Journal

Environment

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples (Video) | JoVE

Méthode EPA 1615. Mesure de entérovirus et Norovirus présence dans l&#39;eau par Culture et RT-qPCR. I. Prélèvement des échantillons de virus

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

Recherche

Environnement

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems (Video) | JoVE

Une approche Cell biorapporteur entières pour évaluer transport et la biodisponibilité des contaminants organiques dans les systèmes insaturés eau

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is ...

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies (Video) | JoVE

L&#39;utilisation de Chironomidae (Diptera) Surface-Flottant nymphose exuvies comme un protocole Rapid Bioassessment des plans d&#39;eau

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR (Video) | JoVE

Méthode EPA 1615. Mesure des entérovirus et Norovirus présence dans l&#39;eau par la culture et RT-qPCR. Partie III. Détection de virus par RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples (Video) | JoVE

Sélection de la taille Gel automatisé pour améliorer la qualité des bibliothèques de séquençage de prochaine génération Préparé à partir d&#39;échantillons d&#39;eau de l&#39;environnement

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants (Video) | JoVE

Une méthode simple pour l&#39;extraction des échantillons d&#39;eau automatisée en phase solide pour immunologique analyse des petits polluants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Air-sampled Filter Analysis for Endotoxins and DNA Content

Air-sampled Filter Analysis for Endotoxins and DNA Content (Video) | JoVE

L&#39;analyse du filtre à air dans l&#39;échantillon d&#39;endotoxines et de l&#39;ADN contenu

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles ...

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity (Video) | JoVE

Préparation et essai d&#39;impédance basée sur biopuces fluidiques avec des cellules RTgill-W1 pour l&#39;évaluation rapide des échantillons d&#39;eau potable pour la toxicité

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples (Video) | JoVE

Méthode d&#39;extraction et d&#39;analyse de lipides pour caractériser les microbes du sol dans les expériences avec de nombreux échantillons

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis (Video) | JoVE

Extraction de pesticides organochlorés à partir de granulés en plastique et analyse de type plastique

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...