This work was funded by the European Union Seventh Framework Program FP7/2007-2013 under grant agreement no. 265721. The authors thank the RIKILT Institute for Food Safety (NL) for their support in this project.

참고 : 다음 프로토콜은 SPE가 50 % v / V를 메탄올로 C18 흡착제 및 용출 100 ml의 물 샘플에서 수행에 대해 설명합니다. 농축 샘플 (ELISA) 키트를 분석 면역 연결 효소 분석하기 전에 10 % v / V를 메탄올에 도달하기 위해 희석한다. 1. 시약 준비 <ol><li> 물 샘플을 준비 <ol><li> 이전의 임의의 다른 단계에, 0.2 ㎛의 공극 크기의 필터를 각각 100 ml의 물 샘플을 필터링. </li><li> 물의 부피 기준으로 용액의 적절한 용적을 희석하여 목적하는 농도를 갖는 샘플 스파이크. 예를 들어, 300 μg의 / L의 농도에서 E2 참조 용액 3.3 μL 희석 E2 100 겨 / L의 물 샘플을 100㎖를 준비한다. 샘플 10 NG / L (E2)와 아군 얻기 위해이 솔루션을 10 번을 희석. 1 NG / L E2와 100 ㎖의 샘플을 얻기 위해 후자의 또 다른 열 배 희석. </li><li> G와 유리 병 (수정되지 않은 또는 아군) 필터 샘플을 배치L45 스레드. 같은 날 샘플을 사용합니다. 작은 부분을 가지고하는 초기 농도를 추정하기 위해 ELISA로 분석된다. </li></ol></li><li> 타이트한 튜브에 50 % v / V를 탈 이온화 (디) 물에 메탄올을 희석하여 리제 300 μl를 준비합니다. </li></ol> SPE 열 2. 준비 <ol><li> 첫번째 메탄올 1,600 μL 후 역상 흡착제의 40 mg의 디 물 400 μl를 첨가하여 옥타 데실 실리카 흡착제 입자의 20 ㎎ / ㎖ 현탁액을 제조. 단단히 뚜껑을 닫고 소용돌이로 교반. </li><li> 열에서 바닥 막 설치 <ol><li> 기공 크기 11 μm의 한 나일론 막을 선택 및 안티 먼지 조직의 더블 레이어에 놓습니다. 3mm 직경의 펀치로, 막에서 두 개의 작은 부분을 절단. </li><li> 평면 엔드 필터 집게와 작은 막 중 하나를 잡고 컬럼의 한쪽에 놓습니다. </li><li> 튜브 평면 하단 커넥터 나사 조이. 멤브레인은 N이고아우 제자리에 안전하게 흡수제 첨가 할 수있다. 막 배치 된 곳 끝으로 열 가리키는 몸에 화살표를 그립니다. </li></ol></li><li> 포장 흡수제 열 준비 <ol><li> 바닥을 향해 가리키는 화살표와 홀더에 열을 고정합니다. 열은 수직으로 가능한 설정해야합니다. </li><li> 루어 락 커넥터를 사용함으로써, 컬럼 튜브의 단부에 빈 10 ㎖를 일회용 주사기를 첨부. </li><li> , 20 ~ 200 μL 팁과 마이크로 피펫을 준비 100 μL에 볼륨을 설정합니다. 이 피펫 팁 포맷이 제조 될 흡착제 컬럼의 크기로 구성된다. 절차는 1 ml의 피펫에 사용되는 것과 같은 큰 피펫 팁을 더 어렵다. </li><li> 소용돌이와 흡착제 서스펜션을 선동하고, 빠르게 열에서 중앙에 100 μl를 피펫. 주입하는 동안, 모든 흡 부드럽게 피펫 팅 용액을 다른 손으로 주사기를 사용하여 멤브레인을 물마루. 본 보안 목표 명세서에서연령, 주사기를 채우는 용액 입자의 명확해야하고, 입자 베드 결국 열에서 관찰 될 수있다. </li><li> 용액에서 입자의 균일 한 현탁액을 보장하기 위해 모든 피펫 팅 단계 사이의 주식 현탁액을 교반하여이 과정을 2 번 이상 반복합니다. 그 결과 열은 흡착제의 6 밀리그램이 포함되어 있습니다. </li><li> 현탁액 300 ㎕를 로딩하고 주사기로 흡입하여 건조되었을 때, 위치에 주사기를 유지하고 다른 손의 집게를 이용하여 상부에 제 나일론 막을 배치했다. </li><li> 튜브와 제 2 커넥터를 나사와 주사기를 폐기하십시오. SPE 열을 사용할 준비가되었습니다. </li></ol></li></ol> 3. 시스템 준비 <ol><li> 루어 잠금 커넥터를 사용하여 장치의 SPE 열을 조입니다. 화살표는 장치에 표시된 것과 같은 방향을 가리키고 있어야한다. </li><li> 나사 결합 장치로 샘플을 함유하는 병을 연결그것에 GL45 안전 캡을 제공했다. </li><li> &#39;리제&#39;저수지에서 리제 200 μl를로드합니다. </li><li> 로드 &#39;희석&#39;저수지에서 디 물 800 μL. </li><li> 추출 단계에서 처리 된 물을 수집하는 폐기물 출구에 병을 놓습니다. 형식은 중요하지 않지만 볼륨 샘플 량에 대해서는 충분히 크게 할 필요가있다. </li><li> 1.5 ml의 최소 볼륨 용량 센서 출구에 작은 유리 병을 놓습니다. 풍부한 출제 및 희석 버퍼를 수집 할 때 거품이 출구에 형성되므로 1 ml의 최대 볼륨이 너무 작은 것입니다. </li><li> 압력 조정기가 수동으로 설정하여 폐쇄 위치에 확인한 역 시계 방향으로 더 이동이 불가능해질 때까지. </li></ol> 프로토 타입 4. SPE <ol><li> 뒷면의 버튼을 눌러 프로토 타입 전환합니다. </li><li> 사용자 인터페이스를 준비하고 프로그램을 선택 <ol><li> 사용자 인터페이스를 엽니 다. 고른컴퓨터의 통신 포트는 장치 목록에서 접속되고, 버튼 &#39;다음&#39;을 클릭하도록한다. </li><li> 값 PSET 580 및 dpset 30을 입력합니다. 펌프는 실행시 580 ± 30 mbar에서 가압 시스템 및 저수지의 압력을 유지하기 위해 조정됩니다. </li><li> 자동 모드를 선택합니다. </li><li> 자동 모드 구석에, 상자 &#39;구성 파일 경로&#39;의 프로그램 파일을로드. </li></ol></li><li> 압력 조정기를 조정 <ol><li> 펌프를 시작합니다. </li><li> 레그에 대한 읽기 값이 열등하지만 320 밀리바에 가까운 때까지 수동 압력 조절기를 돌립니다. </li><li> 펌프를 중지합니다. </li></ol></li><li> SPE의 절차를 시작하기 <ol><li> 자동 모드 모서리를 눌러 &#39;시작&#39;. 펌프에 전환하고 추출, 용출 및 희석 단계는 자동으로 서로를 따를 것이다. 100 ml의 시료에 대한 전체 공정 50 분에서 수행된다. </li><li>레그의 값을 확인합니다. 최적의 유량을 보장하기 위해 추출 공정 동안 350 밀리바 - 그것은 320의 범위에 있어야한다. </li><li> 분석 할 때까지 어둠 속에서 4-5 ° C에있는 작은 유리 병 및 저장을 닫습니다. 분석 물질의 열화를 방지하기 위해 다음 30 시간에서 분석을 수행한다. </li><li> 처리 된 물을 폐기하십시오. </li></ol></li><li> 시스템 청소 주의 : 시스템이 필요한 각 추출 과정은 교차 오염을 방지하기 위해 세정되어야 할 후에. <ol><li> GL (45) 유리 병에 메탄올 70 %의 v / V의 10 ml의 솔루션을 준비합니다. </li><li> SPE 열을 분리 및 커넥터와 튜브를 연결합니다. </li><li> 자동 모드에서, &#39;청소&#39;파일을 선택하고 동일한 압력 설정으로 시작합니다. </li></ol></li></ol> ELISA와 에스트라 디올 농도의 5 검출 <ol><li> 사용되는 ELISA 키트와 함께 제공되는 프로토콜에 표시된 농도 교정 샘플을 준비합니다. 하나 CALIBRA 준비메탄올 10 %의 v / v를 설정 한 것과 같은 샘플로 매트릭스, 한 교정 설정 기. </li><li> 제조업체의 지시에 따라, 플레이트에 웰에서 샘플의 필요한 양을 분배. 교정 샘플을 여과하지만 메탄올 10 % 절에서 샘플을 SPE로 처리하고, 농축되지 않은 수정되지 않은 및 아군 물 샘플을 사용하여 / V. 분석과 관련된 오류를 줄이기 위해 샘플 당 3 우물을 사용합니다. </li><li> 효소와 반응물의 첨가 용 키트, 배양 시간, 세정 및 상기 반응 구비 프로토콜의 표시를 따르. </li><li> 플레이트 리더 악기와 장비 제조업체의 지침에 따라 각 웰에 광 신호를 읽고 데이터를 수집합니다. </li><li> 메탄올 10 % v를 농축 시료를 검량선과 맞 같은 매트릭스와 초기 시료 E2의 농도를 측정하고,하기에서 플레이트 판독기 기기의 소프트웨어를 사용 / V. </li></ol>

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The authors have nothing to disclose.

면역 분석을 사용하여 분석 하였다 물 샘플의 제조를위한 새로운 방법이 제안되었다. 악기가 자동 사용자 친화적 방식으로 고체상 추출을 수행 할 수있다. 종래의 시스템에 주입 물 샘플의 여과는 매우 중요하다. 용액에 존재하는 임의의 미립자는 여전히 잠재적으로 유체 망의 막힘을 일으키고 SPE 칼럼을 방해한다. 다른 중요한 단계는 SPE 칼럼의 제조이다. 열에있는 입자의 ...

샘플은 분석 준비 과정에서 중요한 단계이다. 특히, 매트릭스 효과, 간섭의 감소, 및 분석 물의 농축 제거하여 정확한 결과를 획득하고 검출 하한에 도달하는 데 필요한. 내분비 방해 화합물 (EDCs)은 인해 생물에 대한 자신의 행동에 특별한 관심의 경우에도 환경에서 현재 매우 낮은 수준. 천연 호르몬 17β-에스트라 디올은 유럽 물 기본 지침에 의해 규제되는 우선 순위 물질 목록에 추가 할 수있는 유럽 연합 (EU)의 수질 오염 감시 목록에 현재와 경향이있다. 고체 상 추출 (SPE)는 일반적으로 화학 양 1-5 (크로마토 그래피, 질량 분광 분석) 6-9 및 면역 학적 검출 방법으로, 작은 물 오염 물질의 분석에 적용된다. 후자는 면역이 형식의 대형 다양한에서 사용할 수있는 TA에 고유 한, 환경 모니터링 분야에 대한 관심을 얻었다피검 rget 및 검출 하한에 도달한다. 6, 7, 10, 11, 각종 효소 결합 면역 분석법 (ELISA)은 시판되고 멀티 웰 플레이트에 한 번에 여러 개의 시료를 분석 가능하게한다. 절차는 몇 시간이 걸릴 수있는 연속 반응 단계에있다. 반응의 최종 생성물은 검량선에 기초하여 상기 표적 분자의 농도를 결정하기 위해 광학적으로 검출 할 수있다. 고전 SPE 절차는 용출액의 증발에 의해 흡착 프리 컨디셔닝 (pre-conditioning), 샘플 추출, 세척, 용출 및 농도를 포함한다. 이 추출물을 희석에 사용되는 용제는 검출 방법에 따라 선택된다. 면역 학적 방법에 대해서는, 강하게 유기 용매의 영향에있어서의 감도의 양. 12 회수 및 미리 농도 성능 외에도, 방법은 간단하고 효율적인 비용이 필요하다. procedur의 자동화전자는 인간의 관련 오류를 줄일 수 있습니다. 우리의 이전 연구 13에서 우리는 자동화 된 SPE에 대한 우리의 프로토 타입을 소개하고, 우리의 방법은 해수 시료에서 자연 호르몬 17β-에스트라 디올의 분석에 적용 하였다. 본 동영상으로 우리는 SPE 및 면역 반응에 의해 감지와의 특별한 호환성 기존의 오프라인과 온라인에 비해 우리의 방법의 기술적 인 장점을 강조하고 싶습니다. 우리는 17β-에스트라 디올의 검출 물 샘플에 적용된 프로토콜을 설명한다. SPE는 옥타 데실 실리카 (C18) 흡수제 및 용출 단계가 희석 된 메탄올 행한다 행한다.

<table border="0" cellpadding="0" cellspacing="0" width="609">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Filter membrane 0.2 &#956;m pore size</td>
			<td style="width:107px;">Merck Millipore</td>
			<td style="width:119px;">GNWP04700</td>
			<td style="width:167px;">For sample filtration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nylon membrane 11 &#956;m pore size</td>
			<td style="width:107px;">Merck Millipore</td>
			<td style="width:119px;">NY1104700</td>
			<td>For SPE column</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Disposable biopsy punch 5 mm</td>
			<td style="width:107px;">Medical Budget</td>
			<td style="width:119px;">39302439</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nucleodur C18 ec&#160;</td>
			<td style="width:107px;">Macherey Nagel</td>
			<td style="width:119px;">713550.01</td>
			<td>50 &#956;m particle diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Synthetic sea water</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">SSWS500-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:107px;">VWR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">17beta-estradiol standard</td>
			<td style="width:107px;">Enzo Life Science</td>
			<td style="width:119px;"></td>
			<td>300 ng/ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">17beta-estradiol ELISA kit&#160;</td>
			<td style="width:107px;">Enzo Life Science</td>
			<td style="width:119px;">ADI-900-008</td>
			<td>96 wells, range 30 - 3000 ng/L</td>
		</tr>
	</tbody>
</table>

a simple method for automated solid phase extraction of water samples for immunological analysis of small pollutants

환경 물 샘플의 고체 상 추출 (SPE)을위한 새로운 방법을 제안한다. 개발 된 프로토 타입은 비용 효율적이고 사용자 친화적이며, 신속한 자동화 및 간단한 SPE을 수행 할 수 있습니다. 예비 농축액 낮은 유기 용매 함량, 면역 측정법에 의한 분석과 호환 가능하다. 방법은 100 mL의 물을 샘플에 천연 호르몬 17β-에스트라 디올의 추출 및 미리 농도를 설명한다. 역상 SPE는 옥타 - 실리카 흡착제로 수행되고 용출 메탄올 50 % V / V를 200 μL로 이루어집니다. 용출액은 메탄올의 양을 낮추는 디 물을 첨가하여 희석한다. 수동 SPE 칼럼을 준비한 후, 전체 절차는 1 시간 이내에 자동으로 수행된다. 프로세스의 끝에서, 에스트라 디올 농도 상업용 효소 결합 면역 흡착제 분석법 (ELISA)을 이용하여 측정된다. 100 배 사전 농도를 달성하고 10 %의 V / V에서 메탄올 내용. 분자의 전체 복구1 NG로 달성된다 / L은 드 이온화 및 합성 해수 샘플을 아군.

흡착재 충전부의 재현성이 건조에 의해 평가하고 유리 바이알에 피펫 흡착제 가중치 및 초기 및 사전에도 2. 농도로 나타낸 바와 같이 결과는, 100 mL의 시료에 대해 시험 하였다 주입시의도 1. 재현성에 도시 된 농축 된 샘플은 17β-에스트라위한 상용 ELISA 키트를 사용하여 측정하고,도 3에 도시되어 아군. <p class="jove_content" fo:keep-toget...

Watch this Scientific Journal Video about A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants at JoVE.com

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

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

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 ...

a-simple-method-for-automated-solid-phase-extraction-water-samples

Research

JoVE Journal

Environment

13.6K 조회수.  Centre Suisse d'Electronique et de Microtechnique. A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

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

A Small Volume Procedure for Viral Concentration from Water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

물에서 바이러스 농도에 대한 작은 볼륨 절차

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

연구

환경과학

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

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

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. ...

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

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water&#8217;s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

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

Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

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. ...

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

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

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 ...

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.

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 ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

바다 표면 및 샘플 분석에 Microplastics 샘플링을위한 프로토콜

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

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

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

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

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

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 ...

A Simple Planting Technique for Re-establishing Trees Where Frequent Inundation Occurs

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with herbaceous plants. This protocol describes a simple, cost-effective tree planting technique needed for restoring degraded Everglade tree islands. The design is patterned after a natural Everglades process that creates floating peat islands, which allows tree survival and growth in flooded conditions and often leads to the development of tree islands. Commercially available peat bags were used as the medium for the growth and establishment of potted native tree saplings. The pop-up configuration floated initially and provided additional elevation to minimize inundation, with a single native tree species sapling and a single tree fertilizer spike. During a 3 year study involving 105 pop-ups, most plants survived (80%) and many thrived. Determining whether this technique can establish trees on a degraded tree island will require longer studies and extensive field tests.

This protocol describes a simple, cost-effective tree planting technique for restoring degraded Everglades tree islands that experience inundation. The design creates an island (pop-up) which floats initially and adds elevation to promote tree survival and growth under flooded conditions.

간단한 다시 설정 나무에 대 한 기술을 설치 하 여 자주 침수 발생

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.