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.

NOTA: Questo protocollo non si occupa di metodi di campionamento e di acqua metabarcoding. L&#39;acqua può essere campionato in modi diversi a seconda fini di studio 16 e vedere Miya et al. 12 per i dettagli sui metodi metabarcoding utilizzando primer MiFish. Si noti che l&#39;acqua campione deve essere mantenuto molto freddo e filtrato entro poche ore per evitare la degradazione di Edna. Si noti inoltre che questo protocollo prevede l&#39;utilizzo di un agitatore rotativo e un incubatore...

<ol>
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In molti studi metabarcoding utilizzando campioni ambientali come l&#39;acqua e il suolo, il trattamento post-filtrazione della cartuccia filtrante è generalmente come segue 24,25: 1) taglio aperto e screpolature della custodia con utensili manuali (taglio per tubi o pinze); 2) la rimozione del filtro dalla cartuccia; e 3) taglio del filtro in piccoli pezzi con una lametta per l&#39;estrazione del DNA. Per evitare tali diversi metodi di estrazione del DNA ingombrante e richiede tempo procedure che sono sogge...

DNA ambientale (Edna) in ambienti acquatici si riferisce a materiale genetico trovato nella colonna d&#39;acqua. Recenti studi hanno dimostrato l&#39;utilità di Edna per la rilevazione di pesci da vari ambienti acquatici, tra stagni, fiumi 1-3 4-8, torrenti 9, e l&#39;acqua di mare 10-14. La maggior parte di questi studi si sono concentrati sulla individuazione di un unico o pochi invasivo 1,4-6,8,14 e rare o minacciate specie di 3,9, mentre alcuni studi recenti hanno tentato il rilevamento simultaneo di più specie in comunità ittiche locali 7,9, 12,13,15 e mesocosmi 11,12. Quest&#39;ultimo approccio è chiamato &quot;metabarcoding&quot; e Edna metabarcoding utilizza uno o più set di primer PCR per coamplify una regione del gene attraverso campioni tassonomicamente diversi. Questo è seguito da preparazione biblioteca indicizzazione e aggiunta adattatore, e le librerie indicizzati sono analizzate per un high-throughput sequenziamento parallelopiattaforma. Recentemente Miya et al. 12 hanno sviluppato universali primer PCR per metabarcoding Edna dai pesci (chiamati &quot;MiFish&quot;). I primer MiFish bersaglio una regione ipervariabile della mitocondriale 12S rRNA gene (163-185 bp), che contiene informazioni sufficienti per identificare i pesci alla famiglia tassonomica, genere e specie ad eccezione di alcuni congeneri strettamente correlati. Con l&#39;uso di questi primer a Edna metabarcoding, Miya et al. 12 rilevati più di 230 specie marine subtropicali da acquari con noti composizione delle specie e barriere coralline vicino l&#39;acquario. Mentre l&#39;ottimizzazione del protocollo metabarcoding per accogliere l&#39;acqua di mare naturale, con livelli di concentrazione Edna dai pesci varia, abbiamo notato che i primer MiFish di tanto in tanto non è riuscito a amplificare la regione di destinazione per la successiva preparazione biblioteca. Uno dei motivi più probabili per questo l&#39;amplificazione PCR infruttuoso è la mancanza di adeguate quantità di Template DNA contenuto in piccoli volumi di acqua filtrata (cioè 1-2 L). Anche se la concentrazione Edna da uno specifico gruppo tassonomico è inconoscibile prima l&#39;amplificazione, la filtrazione di grandi volumi d&#39;acqua (&gt; 1-2 L) sarebbe un mezzo semplice ed efficace per raccogliere più Edna dagli ambienti acquatici con scarsa abbondanza di pesce e la biomassa, come ad esempio open-mare e in acque profonde ecosistemi. Rispetto al filtri in fibra disco convenzionalmente utilizzato in un certo numero di ricerca pesci Edna 16, cartucce filtranti hanno il vantaggio di ospitare volumi idrici grandi prima intasamento 17. In realtà, un recente studio ha dimostrato grande volume (&gt; 20 L) filtrazione di campioni di acqua di mare costiere che utilizzano cartucce filtranti 18. Inoltre, essi sono confezionati singolarmente e sterile, e diversi passaggi del flusso di lavoro sperimentale possono essere eseguite nell&#39;alloggiamento del filtro, riducendo così la probabilità di contaminazione da laboratorio 19. L&#39;ultimocaratteristica è fondamentale per Edna metabarcoding, in cui il rischio di contaminazione rimane tra le più grandi sfide sperimentale 20,21. Nonostante questi vantaggi tecnici di cartucce filtranti, non è stato utilizzato in studi Edna di pesci con due eccezioni 8,15. Qui forniamo un protocollo per la filtrazione dei campioni di acqua con la cartuccia del filtro e l&#39;estrazione di Edna dal filtro senza dover tagliare aprire l&#39;alloggiamento. Forniamo anche due sistemi di filtrazione dell&#39;acqua alternativi a seconda dei volumi d&#39;acqua (≤4 L o&gt; 4 L). Per confrontare le prestazioni del protocollo di nuova concezione e un protocollo precedentemente utilizzato utilizzando un filtro in fibra di vetro nel nostro gruppo di ricerca 12,14,22,23, eseguiamo Edna metabarcoding analisi di acqua di mare da un serbatoio acquario enorme (7.500 m 3 ) con nota composizione delle specie, e visualizza il numero di specie rilevate derivati ​​dai due protocolli come risultati rappresentativi. Questo protocollo hcome stato sviluppato per la metabarcoding Edna dai pesci, ma è anche applicabile ad Edna da altri organismi.

<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.

È tecnicamente difficile isolare e quantificare solo Edna pesce dal Edna massa estratta, perché i primer MiFish coamplify la regione di destinazione da parte di alcuni vertebrati non-pesce, come uccelli e mammiferi, con prodotti di PCR della stessa dimensione (ca. 170 bp ) 12. Invece di quantificare pesce Edna, eseguiamo MiFish metabarcoding analisi di Edna da un serbatoio acquario con nota la composizione delle specie utilizzando i due diversi metodi di filtraggio ...

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

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.

L&#39;utilizzo di una cartuccia filtrante per filtrazione di acqua campioni ed estrazione di DNA ambientale

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

The overall goal of this protocol is to filter water samples with a filter cartridge and extract environmental DNA without having to cut open the housing to remove the filter from metabarcoding eDNA from various aquatic organisms. This method can help answer key questions in the ecological field, such as biodiversity monitoring and community ecology. The main advantage of this technique is that the filter cartridges can accommodate larger water volumes and the filtration and DNA extraction can be performed in a sterile manner.To begin this procedure, attach high vacuum tubing to the input connector of an aspirator pump. Attach the other end of the tubing to a manifold. Be sure that the three red T-valves at the manifold are in the off position.Next, put on a clean set of gloves. Insert a female luer fitting at the top end of the vacuum rubber tubing. Then, insert a vacuum connector at the bottom end.Carefully attach the female luer fitting to an outlet port of the filter cartridge. Then, attach the vacuum connector to a silicone stopper. Pour one liter of sampled water into the prepared plastic bag.Enclose the screw cap with the male luer-lock connector. Then, carefully connect an inlet port of the assembled filter cartridge with the male luer-lock connector. Ensure that all connections are secure.Then, hang the plastic bag from two prongs on the mesh panel. Insert a silicone stopper into the inlet port of the manifold. Then, turn on the aspirator.Open the red T-valves for filtration. Run the manifold until the filter cartridge is dry. Then, close the red T-valves.Repeat this entire process to filter the desired amount of water. Once all of the sampled water has been filtered, carefully remove the filter cartridge from the plastic bag and vacuum container. Cap both ends of the cartridge with the appropriate luer caps.Then, using a solvent proof pen, label the cartridge and inlet luer cap. Store at 20 degrees Celsius until ready to perform DNA extraction. For samples larger than four liters, prepare a ten liter book bottle as detailed in the text protocol.Add the water sample to be filtered. Then, tightly insert a ten milliliter pipette tip into the valve of the book bottle. Tightly insert the inlet port of the filter cartridge into the pipette tip.Then, insert a silicone stopper into the inlet port of the manifold. Turn on the aspirator. Then, open the red T-valves for filtration.Run the manifold until the filter cartridge is dry. After the filtration is complete, close the red T-valves. Carefully remove the cartridge from the plastic bag and vacuum connector.Then cap both ends of the cartridge with the appropriate luer caps. Use a solvent proof pen to label the cartridge and the inlet luer cap. Store at 20 degrees Celsius until ready to perform DNA extraction.Preheat an incubator to 56 degrees Celsius. Then, cut the hinged cap from a 2.0 milliliter tube and discard the cap. Remove the inlet luer cap from the filter cartridge.Then, insert the inlet port into the 2.0 milliliter tube. Using a self-sealing film, tightly seal the connection between the cartridge and the collection tube. Next, insert the combined unit into a centrifuge adapter for a 15 milliliter conical tube.Centrifuge the cartridge for one minute at 5000 times g to remove the remaining liquid in the filter. Once centrifugation is complete, remove the collection tube from the filter cartridge and discard the tube. Then, recap the inlet port.Next, mix 20 microliters of protein A-Case solution, 220 microliters of phosphate-buffered saline, and 200 microliters of buffer AL.Remove the inlet cap and add the mixture into the cartridge using a pipette, inserting the pipette completely into the inlet port. Recap the inlet port. Then, place the filter cartridge on a rotary shaker.Place the rotary shaker in the preheated incubator, and set the shaker to 20 rpm for 20 minutes. During the incubation, label a new 2.0 milliliter tube with a solvent proof pen. Then, place it into a 50 milliliter conical tube.After the incubation is complete, remove the inlet cap. Then, insert the inlet port into the new 2.0 milliliter tube. Cap the conical tube with a screw cap.Centrifuge for one minute at 5000 times g to collect the extracted DNA. Then, remove the cartridge in the 2.0 milliliter tube using sterilized forceps. Cap the tube and purify the extracted DNA as outlined in the text protocol.In this procedure, four different volumes of sea water are filtered on the same manifold using two unique filters, and eDNA is subsequently extracted. The filter cartridge detected, on average, 50%more species than the glass fiber filters. Once mastered, this technique can be done in two hours if it is performed properly.After watching this video, you should have a good understanding of how to filter water samples with a filter cartridge, and how to extract eDNA without having to cut open the housing to remove the filter from metabarcoding eDNA from various aquatic organisms.

Research

JoVE Journal

Environment

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

Metodo EPA 1615. Misura di Enterovirus e Norovirus Evento in acqua per la cultura e RT-qPCR. I. Raccolta di campioni di virus

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

Ricerca

Ambiente

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

A Whole marcatura già cellulare approccio per valutare i trasporti e la biodisponibilità di contaminanti organici in acqua insaturi Sistemi

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

Uso di Chironomidae (Ditteri) pupa esuvie come Rapid Bioassessment protocollo per i corpi idrici-galleggiante Surface

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

Metodo EPA 1615. Misura di Enterovirus e Norovirus Presenza in Water di Cultura e RT-qPCR. Parte III. Rilevamento virus mediante 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 Selection Size per migliorare la qualità delle Biblioteche di sequenziamento di nuova generazione Preparato da campioni di acqua ambientale

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

Un metodo semplice per Automated fase solida estrazione di campioni di acqua per l&#39;analisi immunologiche di piccole inquinanti

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

Filtro Analisi aria-campionato per endotossine e contenuto di DNA

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

Soil Lysimeter Excavation for Coupled Hydrological, Geochemical, and Microbiological Investigations

Terreno lisimetro scavo per accoppiate idrologici, geochimiche e microbiologiche Investigations

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled ...

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

Un metodo di estrazione e analisi lipidica per la caratterizzazione dei microbi del suolo negli esperimenti con molti campioni

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

Estrazione di pesticidi di organochlorato da pellicole di plastica e analisi di tipo plastico

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