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.

참고 :이 프로토콜은 물 샘플링 및 metabarcoding 방법과 거래를하지 않습니다. 물은 연구 목적 (16)에 따라 다른 방식으로 샘플링 볼 미야 등. (12) MiFish 프라이머를 사용하여 metabarcoding 방법에 대한 자세한 내용은 될 수있다. 샘플링 된 물이 매우 추운 유지와 에드나의 저하를 방지하기 위해 몇 시간 내에 필터링해야합니다. 또한,이 프로토콜은 회전 진탕 배양기와의 사용을 포함...

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물, 토양 등의 환경 시료를 사용하여 많은 metabarcoding 연구를 다음과 같이, 필터 카트리지의 후 여과 처리는 일반적으로 24, 25 : 1)로 개방 절단 또는 손 도구 (튜브 커터 치)와 하우징 분해; 2) 필터 카트리지에서 제거; 3) DNA 추출을위한 면도날 작은 조각으로 절단 필터. 널리 사용되는 저가의 상용 키트를 사용하여 필터 카트리지의 하우징 내에 이러한 귀찮고 시간 소모적 인 실험실 오염되기...

수생 환경에서 환경 DNA (에드나)는 물 칼럼에서 발견 된 유전 물질을 의미한다. 최근의 연구는, 연못 1-3, 강, 4-8 등 다양한 수생 환경에서 물고기를 검출하는 에드나의 유용성을 입증 9 스트림, 해수 10-14. 최근의 연구는 생선 커뮤니티 7,9에서 여러 종의 동시 검출을 시도하면서 이러한 연구의 대부분은, 3,9 종 단일 또는 소수의 침략 1,4-6,8,14 희귀 또는 위협의 탐지에 초점을 맞춘 12,13,15 및 반 폐쇄 생태계 (11, 12). 후자의 방법은 &quot;metabarcoding&quot;라고하며 에드나의 metabarcoding은 분류 학적으로 다양한 샘플에서 유전자 영역을 coamplify하는 PCR 프라이머의 하나 또는 여러 개의 세트를 사용합니다. 이것은 인덱싱 및 어댑터 첨가 라이브러리를 제조 하였다되고 인덱싱 라이브러리는 높은 처리량 병렬 염기 서열 분석플랫폼. 최근 미야 등. (12) ( &quot;MiFish&quot;라고합니다) 물고기에서 에드나를 metabarcoding에 대한 보편적 인 PCR 프라이머를 개발했다. MiFish 프라이머는 분류 학적 가족, 속 일부 밀접하게 관련 동족체를 제외하고 종 물고기를 식별하는 데 충분한 정보가 들어있는 미토콘드리아 12S rRNA 유전자 (163-185 BP)의 가변 영역을 대상으로. 에드나의 metabarcoding에서와 프라이머를 사용하여, 미야 등. (12)는 수족관 근처 알려진 종 조성과 산호초 수족관 탱크에서 230 개 이상의 아열대 해양 종을 발견했습니다. metabarcoding 프로토콜을 최적화하는 물고기에서 에드나 농도 다양한 수준의 천연 해수를 수용 할 수 있지만, 우리는 MiFish 프라이머 가끔 후속 라이브러리 준비를위한 대상 영역을 증폭하지 못한 것으로 나타났습니다. 이 실패한 PCR 증폭 용 가능성 이유 중 하나는 TE 충분한 양의 부족여과 물 소량에 포함 mplate DNA (즉, 1-2 L). 특정 분류 학적 그룹에서 에드나 농도가 큰 물 볼륨 (&gt; 1-2 L)의 여과가 증폭되기 전에 알 수없는하지만 것 희귀 물고기의 풍부한 바이오 매스, 같은과 수생 환경에서 더 많은 에드나를 수집 할 수있는 간단하고 효과적인 수단이 될 오픈 바다와 심해 생태계. 디스크 섬유 필터에 대하여 종래에 물고기 에드나 연구 (16)의 수에 사용 된 필터 카트리지 (17)를 폐색하기 전에 물 큰 볼륨을 수용하는 장점을 갖는다. 사실, 최근의 연구는 많은 양의 (&gt; 20 L) 필터 카트리지 (18)를 사용하여 연안 해수 시료의 여과를 보였다. 또한, 이들은 개별 포장 및 멸균하여 실험 워크 플로우의 여러 단계가 이에 해당 기관 (19)로부터의 오염 가능성을 감소시키는 필터 하우징에서 수행 될 수있다. 후자의기능은 가장 큰 실험은 20, 21에 도전 중있는 오염의 위험이 남아 에드나의 metabarcoding 중요합니다. 필터 카트리지의 이러한 기술적 장점에도 불구하고, 그것은 두 가지 예외 8,15와 물고기 에드나 연구에 사용되지 않았다. 여기에서 우리는 주택을 열 절단 할 필요없이 필터에서 에드나의 필터 카트리지 및 추출 물 샘플의 여과를위한 프로토콜을 제공합니다. 우리는 또한 물 볼륨 (≤4의 L 또는&gt; 4 L)에 따라 두 가지 대체 물 ​​여과 시스템을 제공합니다. 새로 개발 된 프로토콜의 성능과 우리의 연구 그룹 12,14,22,23에서 유리 섬유 필터를 사용하여 이전에 사용되는 프로토콜을 비교하기 위해, 우리는 에드나는 거대한 수족관 탱크에서 해수의 분석을 metabarcoding 실시합니다 (7,500m 3 ) 알려진 종 조성물과 대표적인 결과로서 두 프로토콜로부터 유도 검출 종의 수를 나타낸다. 이 프로토콜의 시간으로는 물고기에서 에드나를 metabarcoding 위해 개발하지만, 다른 생물체에서 에드나에도 적용되었다.

<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.

그것은 MiFish 프라이머는 조류와 포유류와 같은 일부 비 물고기 척추 동물에서 대상 지역을 coamplify 때문에 같은 크기의 PCR 제품, 분리 및 추출 된 대량 에드나 만 물고기 에드나를 정량화하는 것은 기술적으로 어렵다 (캘리포니아. 170 bp의 ) 12. 대신 물고기 에드나를 정량화, 우리는 여과 및 DNA 추출의 두 가지 방법을 사용하여 알려진 종 구성 수족관 탱크에서...

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.

여과 물 샘플 및 환경 DNA의 추출을위한 필터 카트리지의 사용

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

엔테로 바이러스 및 문화 RT-qPCR에 의해 물에서 노로 바이러스의 발생의 EPA 방법 1615 측정. 바이러스 샘플의 I. 컬렉션

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

연구

환경과학

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

전체 셀 Bioreporter 접근 물 불포화 시스템에서 유기 오염 물질의 운송과 생체 이용률을 평가하기 위해

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

Chironomidae (파리목)의 사용 표면 부동 물 기관에 대한 신속한 생물학적 평가 프로토콜로 번데기 Exuviae을

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

엔테로 바이러스 및 문화와 RT-qPCR에 의해 물에서 노로 바이러스의 발생의 EPA 방법 1615 측정. 파트 III. RT-qPCR에 의한 바이러스 검출

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

자동 젤 사이즈 선택은 환경 물 샘플에서 준비 차세대 시퀀싱 라이브러리의 질을 향상시키기 위해

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

작은 오염 물질의 면역 학적 분석을위한 물 샘플의 자동화 된 고체상 추출을위한 간단한 방법

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

독소와 DNA 내용에 대한 공기 샘플링 필터 분석

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

준비 및 시험 독성에 대한 신속한 평가 음주의 물 샘플에 대한 RTgill-W1 세포와 유체 바이오칩을 임피던스 기반

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

많은 샘플을 이용한 실험에서 토양 미생물을 특성화하기위한 지질 추출 및 분석 방법

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

플라스틱 펠렛 및 플라스틱 유형 분석에서 유기 염소 농약의 추출

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