이 작품은 농업 건강과 안전 (5U54OH008085)에 대 한 CDC/NIOSH 높은 평야 Intermountain 센터와 콜로라도 생명 과학 발견 평가 그랜트 프로그램 (14BGF-16)에서 자금에 의해 지원 되었다.

1. Bioaerosol 챔버의 설치 ( 그림 2참조)
<ol><li>
사전에 0.01 M 버퍼링 인산 염 분, pH 7.5 (PBS)의 20 mL와 함께 액체 impingers 로드 합니다.
	<ol>
<li>음이온 교환 수 지의 0.5 g을 추가 하 고 컨트롤 역할을 수행 하는 다른 액체 임 핀 저와 액체 impingers 중의 PBS 내 일시 중단.</li>
	</ol>
</li>
	<li>챔버 내부에 bioaerosol는 분무기를 직면 하는 어로 졸 후미와 클램프 스탠드를 사용 하 여 병렬로 위치 액체 impingers. 참고: 추가 정보에 대 한 그림 2 를 참조 하십시오.</li>
	<li>공동 직접 읽기 졸 모니터를 bioaerosol의 대량 농도 (mg/m3)을 측정 하 여 액체 impingers 가까이 찾습니다.</li>
	<li>추가적인 직접 읽기 계측 (실내 공기 질 모니터 및 열 풍속 계) 배치, 온도 등의 환경 변수를 모니터링 하는 액체 impingers 근처 상대 습도 바람 속도, 챔버 내에 bioaerosol.</li>
	<li>에 어로 졸의 혼합을 촉진 하기 위하여 bioaerosol 챔버의 모서리에 축 팬 들을 실행 합니다. 참고: 축 팬 고정 속도로 실행 하 고는 조정 가능 하다.</li>
</ol>2. 모델 Bioaerosolization 실험 ( 그림 3참조)
<ol><li>시리얼, 인산 염 버퍼 식 염 수 (PBS)에서 원하는 농도 MS2 살 균 소 및 인플루엔자 백신의 10 희석을 수행 합니다. 참고: 여기, 103.5 : FFU/mL는 종류 A와 B 독감 바이러스 백신 긴장의와 10 메서드를 보여 주기 위해5 PFU/mL MS2 살 균 소의 활용 됩니다. Titers는 inocula에 대 한 결정, 살 균 소는 대장균 HS (pFamp) R에에서 전파 되며 앞에서 설명한20으로 열거. 상용 백신 포함 알려진된 양의 4 라이브 감쇠 독감 바이러스 변종, 두 입력 인플루엔자 바이러스와 형광 초점 단위 또는: FFU 표현 두 B 형 인플루엔자 바이러스.</li>
	<li>100 mL PBS의에서 바이러스 성 입자의 원하는 농도의 정지를 만들어서 6 제트기 충돌 분무기의 유리 용기 내에서 inocula를 준비 하 고 사용자 지정 bioaerosol 챔버 내 설치.</li>
	<li>계측 변수 챔버 내에 bioaerosol, 온도, % 상대 습도, 이산화탄소 농도 등을 측정 하 고 바람 속도 (축 팬을 사용 하 여 생성)을 시작 합니다. 직접 읽기 졸 모니터를 사용 하 여 챔버 내에 bioaerosol 질량 농도 추적 하 고 시간과 실험 사이 일관성을 확립.</li>
	<li>Bioaerosol 챔버를 밀봉 하 고 공기 HEPA 필터와 10 분 동안 제거.</li>
	<li>6.9 kPa에서 분무기에 필터링 된, 건조 공기를 제공 합니다.</li>
	<li>각 bioaerosol 챔버 시험 (nebulizer)를 통해 바이러스 bioaerosols의 대상 농도 생성 합니다. 이 데모 5 mg/m3 의 농도와 bioaerosols 생성 됩니다.</li>
	<li>에 어로 졸의 대상 질량 농도 도달 하면 일단 nebulization을 중지 합니다.</li>
	<li>사용 하 여 기본 흐름 표준 각 샘플링 이벤트 전 / 후 볼륨 샘플링에 일관성을 보장 하는 행위 교정 12.5 L/분의 유량을 조정 되었습니다 각 액체 임 핀 저에 대 한 공기 샘플링 펌프를 작동 합니다. 공급 희석 HEPA 필터 라인 부근에 분무기를 사용 하 여 공기와 챔버에 일정 한 압력을 유지.</li>
	<li>적극적으로 샘플 당 500 L의 샘플링 볼륨을 달성 하기 위해 40 분의 기간에 대 한 bioaerosol 챔버 분위기를 샘플 합니다. 참고: 동시에 두 개의 펌프와 12.5 L/분 40 분에 대 한 샘플링은 bioaerosol의 1000 L의 샘플링에 대 한 허용 됩니다. 따라서, 실험의 완료에 예상 된다 bioaerosol 약 실에 있는 공기의 대부분을 샘플링 하 고 HEPA 필터 시스템을 통해 환경으로 출시 되었습니다. 이 입자 농도 감소에 의해 입증 됩니다. 후 실험 표면 10% 가구 표 백제와 70% 에탄올으로 소독 된다. 표시기;이 데모에 사용 됩니다. 그러나, 알려진 병원 성 유기 체는이 시스템에 사용 됩니다, 추가 biosafety 조치 수 있습니다 구현 필요에 따라.</li>
</ol>3. 핵 산 추출 및 qRT-PCR 검출
<ol><li>
음이온 교환 수 지에서 직접 하 고, 액체 impingers 내에서 사용 하는 액체에서 비교 목적을 위해 핵 산을 분리. 이 예제에서는 RNA 격리 절차 설명 하지만 비슷한 프로토콜 DNA 바이러스를 위해 사용 될 수 있습니다.
	<ol>
<li>음이온 교환 수 지에서 핵 산 격리입니다.<ol>
<li>살 균 50 mL 원뿔 튜브에 음이온 교환 수 지를 포함 하는 액체의 액체 임 핀 저의 모두를 전송.</li>
			<li>정착, 수 지 고 천천히 음이온 교환 수 지에서 액체 샘플을 가만히 따르다. 1 mL 피 펫 팁을 사용 하 여 음이온 교환 수 지에서 남은 액체의 모든을 제거.</li>
			<li>바이러스 성 RNA 격리 키트에서 포함 하는 음이온 교환 수 지에 직접 캐리어 RNA 바이러스 성 세포의 용 해 버퍼의 560 µ L을 추가 하 고 정기적으로 혼합과 실 온에서 10 분 동안 품 어.</li>
			<li>1 mL 피 펫 팁을 사용 하는 바이러스 성 전송 lysate (바이러스 성 세포의 용 해 버퍼) 살 균 1.5 ml 원뿔 튜브와 RNA 계속 바이러스 성 RNA 격리를 사용 하 여 격리 키트 ( 재료의 표참조) 제조업체의 지침에 따라 제외 하 고 그 RNA는 eluted 차입 버퍼의 60 µ L의 전체 볼륨에.</li>
		</ol>
</li>
		<li>핵 산 격리 액체 impingers에서.<ol>
<li>살 균 1.5 mL 원뿔 튜브에 액체 샘플의 140 µ L 플라스틱 고 RNA 격리 차입 버퍼의 60 µ L의 전체 볼륨에 RNA를 방출 하는 것 제외 하 고 제조업체의 지침에 따라 바이러스 성 RNA 격리 키트를 사용 하 여 수행 합니다. 참고: 140 µ L은 단일 단계 준비에서 여기 고용 특정 바이러스 성 RNA 격리 키트와 함께 처리할 수 있는 최대 샘플 볼륨입니다.</li>
		</ol>
</li>
	</ol>
</li>
	<li>앞에서 설명한23,24MS2 및 인플루엔자 A와 B 바이러스의 검출을 위한 qRT-PCR을 수행 합니다. 이 데모에서는 핵 산 있다의 5 µ L qRT-PCR에 사용 됩니다.</li>
</ol>

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J., Martel, B., Moineau, S., Duchaine, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+five+bacteriophages+as+models+for+viral+aerosol+studies.">Comparison of five bacteriophages as models for viral aerosol studies.</a> Applied and Environmental Microbiology. 80 (14), 4242-4250 (2014).</li><li>Vergara, G. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+FRNA+coliphages+as+indicators+of+human+enteric+viruses+in+a+tropical+urban+freshwater+catchment.">Evaluation of FRNA coliphages as indicators of human enteric viruses in a tropical urban freshwater catchment.</a> Water Research. 79, 39-47 (2015).</li><li>Tung-Thompson, G., Libera, D. A., Koch, K. L., de Los Reyes, F. L., Jaykus, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerosolization+of+a+human+norovirus+surrogate,+bacteriophage+MS2,+during+simulated+vomiting.">Aerosolization of a human norovirus surrogate, bacteriophage MS2, during simulated vomiting.</a> PLoS One. 10 (8), e0134277 (2015).</li></ol>

저자는 공개 없다.

이 프로토콜에서 bioaerosols 수정된 액체 impingers를 사용 하 여 민감한 바이러스 캡처 하는 방법을 설명 합니다. 방법은 탐지 및 정량화 bioaerosols에 바이러스 부하의 최적화 되어 있습니다. 여기 설명 하는 특정 수정 일반적인 액체 임 핀 저 안에 포함 된 액체에 음이온 교환 수 지의 추가 포함 한다. 이 메서드는 원심 분리, 여과, 강 수 기반 방법 등 다른 샘플 처리 기술 이러한 이점을 제공 하지 않는 ?...

이 방법의 목적은 bioaerosols에서 부정 청구 바이러스의 분자 탐지를 촉진 하기 위하여 매우 중요 한 bioaerosol 샘플링 플랫폼을 제공. 미생물, 바이러스 성 입자를 포함 하 여 장시간 시간1bioaerosols에서 살아남을 수 있습니다. Bioaerosols 비교적 긴 거리를 여행 하 고 생존 및 infectivity, 산업 영향 받은 개인에서 6 킬로미터의 거리에 있는 타워에서 Legionnaires' 질병의 발발에 의해 입증으로 유지 수 및 18 사망자2결과. 간접 전송 bioaerosols 중재 하는 인 간에 게 바이러스의 여러 설정에서 발생할 수 있으며 학교와 레스토랑3,4노로바이러스 감염에 대 한 입증 되었습니다. 바이러스의 bioaerosol 전송의 생산 시설5, 바이러스의 운동에 주요 요인으로 간주 되 고이 전송 경로와 마찬가지로, 돼지 및 가금류 농장에서와 같은 농업 설정에서 발생할 수 있습니다. 6 , 7 , 8 , 9.
시위는 H5 독감 바이러스는 살아있는 동물에서 bioaerosols에서 발견 된 중국에서 시장 에서처럼 바이러스 라덴 bioaerosols의 효과적인 샘플링 신속한 진단 및 확산 방지를 위한 대비에 개선을 위한 수 고는 미국10,11. 현재 bioaerosol 샘플링 기술을 다른 입자 캡처 원칙의 숫자를 포함 하 고 impingers, 사이 클론, impactors, 및 필터12에 광범위 하 게 분류 될 수 있다. 그것은 철저 하 게 모든 장점을 커버 하이 프로토콜의 범위와 bioaerosols;에서 바이러스의 샘플링에 대 한 이러한 플랫폼의 단점 그러나, 그것은 바이러스 및 살 균 소13의 컬렉션에 대 한 이러한 샘플링 장치의 대다수는 최적화 되지 진술 될 수 있다. 또한, 바이러스 성 입자의 infectivity은 종종 부정적인 영향을, 액체 impingers 바이러스 infectivity 샘플링 장치 고체 impactors 또는 필터14보다 더 효과적으로 유지 하는 것으로 간주와 함께. 그러나, 하나의 액체 충돌의 단점은 대상 희석 효과, 바이러스는 비교적 많은 양의 (일반적으로 ≥20 mL) 액체 수집 용기에 수집 때문에 발생 합니다. 또 다른 중요 한 단점은 집중 입자를 액체 impingers의 차선의 효율성 포함 < 0.5 µ M 크기15. 그러나, 이러한 장치의 캡처 효율 immobilization 바이러스 성 핵 산 바이러스 infectivity16,17의 보존을 향상 시킬 수 있습니다으로 고체 행렬에 동원 정지에 의해 개선할 수 있습니다.
우리는 이전 음이온 교환 수 지 캡처 및 F-RNA 살 균 소, A 형 간염 바이러스, 인간 아 데 노비 루스, 고로18,19 포함 하 여 액체 매트릭스에서 바이러스의 농도 대 한 효과적인 도구입니다 증명 하고있다 ,20. 제조 업체에 의해 정의 된 대로이 작품에서 음이온 교환 수 지는 기능성된 4 급 아민 그룹 중재 하십시오 매력 및 액체 매체21 에서 음이온의 캡처 macroreticular 폴리스 티 렌 강한 기본 음이온 교환 수 지 . 따라서, 음이온 교환 수 지 그물 네거티브 표면 요금, 많은 장 바이러스, 인플루엔자 바이러스 및 다른 바이러스 관련 공공 및 동물 건강을 포함 하 여 바이러스를 캡처 예정 이다.
현재 프로토콜 액체 임 핀 저를 음이온 교환 수 지의 추가 포함 한다. 이 시스템에서 수 지 임 핀 저 액체에서 바이러스 성 입자에 대 한 보조 농도 단계 역할을 합니다. 핵 산 분자 분석에 대 한 집중된 샘플을 제공 하는 작은 볼륨에서 직접 eluted 수 다음. 따라서,이 방법의 주요 장점은 주로 샘플 볼륨 감소를 통해 바이러스 검출 감도에 개선 이다. 또한, 때문에 부정적인 충전 바이러스의 고유의 일반적인 캡처, 방법은 가능성이 많은 관심의 바이러스의 탐지를 위해 적용 가능한. 여기, 메서드는 백신 긴장의 유형 A와 B 형 인플루엔자 바이러스와 FRNA coliphage MS2 (MS2)에 대 한 보여 줍니다. 이러한 바이러스 그 후 앞에서 설명한22로 표준 qRT-PCR 분석 실험을 사용 하 여 검색 됩니다. 끝점 사용자 때문에이 메서드를 수행에 문제가 발생할 것을 기대 하지 해야 수정 현재 기존 장비 bioaerosol 샘플링 및 분석의 기존 흐름에 주요 중단을 구성 하지 않습니다.

<table>
	<tbody>
		<tr>
			<td>Escherichia coli bacteriophage MS2 (ATCC 15597-B1)</td>
			<td>American Type Culture Collection</td>
			<td>ATCC 15597-B1</td>
			<td></td>
		</tr>
		<tr>
			<td>FluMist Quadrivalent</td>
			<td>AstraZeneca</td>
			<td>Contact manufacturer</td>
			<td>Viral constitutents of this vaccine are subject to change on an annual basis</td>
		</tr>
		<tr>
			<td>CFX96 Touch Real-Time PCR Detection System</td>
			<td>Bio-Rad</td>
			<td>1855195</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers and probes</td>
			<td>Integrated DNA Technologies</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 &#181;M sterile filter</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>1 L pyrex bottles or equivalent</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL pipet tips</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL pipettor</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL serological pipet</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-aid or equivalent</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp Viral RNA Mini Kit</td>
			<td>Qiagen</td>
			<td>52904</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiTect Probe RT-PCR Kit</td>
			<td>Qiagen</td>
			<td>204443</td>
			<td></td>
		</tr>
		<tr>
			<td>Amberlite IRA-900 chloride form</td>
			<td>Sigma-Aldrich</td>
			<td>216585-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P5368-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Water (molecular biology grade)</td>
			<td>Sigma-Aldrich</td>
			<td>W4502-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf DNA LoBind Microcentrifuge Tubes</td>
			<td>ThermoFisher</td>
			<td>13-698-791</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL Conical Centrifuge Tubes&#160;</td>
			<td>ThermoFisher</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Polypropylene Centrifuge Tubes</td>
			<td>ThermoFisher</td>
			<td>05-538-62</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript III Platinum One-Step qRT-PCR Kit w/ROX</td>
			<td>ThermoFisher</td>
			<td>11745100</td>
			<td></td>
		</tr>
		<tr>
			<td>SKC Biosampler 20 mL, 3-piece glass set</td>
			<td>SKC Inc.</td>
			<td>225-9593</td>
			<td></td>
		</tr>
		<tr>
			<td>Vac-u-Go sample pumps</td>
			<td>SKC Inc.</td>
			<td>228-9695</td>
			<td></td>
		</tr>
		<tr>
			<td>Collison nebulizer (6-jet)</td>
			<td>BGI Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPA capsule</td>
			<td>PALL</td>
			<td>12144</td>
			<td></td>
		</tr>
		<tr>
			<td>Q-TRAK indoor air quality monitor 8554</td>
			<td>TSI Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Alnor velometer thermal anemometer AVM440-A</td>
			<td>TSI Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>SidePak AM510 personal aerosol monitor</td>
			<td>TSI Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioaerosol chamber</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of viruses from bioaerosols using anion exchange resin

이 프로토콜에서는 바이러스에 대 한 사용자 지정된 bioaerosol 샘플링 방법을 보여 줍니다. 이 시스템에서는, 음이온 교환 수 지에서 bioaerosols 부정 청구 바이러스의 효능 농도 액체 치는 행위 기반 공기 샘플링 디바이스와 결합 이다. 따라서, 수 지 bioaerosol 샘플링 워크플로에 추가 농도 단계 역할을 합니다. 바이러스 성 입자의 핵 산 추출 다음 결과 샘플 분자 분석에 대 한 적합 한 음이온 교환 수 지에서 직접 수행 됩니다. 또한,이 프로토콜 사용자 bioaerosol 챔버 다양 한 환경 조건과 환경 변수 온도, 습도, 등의 지속적인 모니터링에 대 한 허용 아래 바이러스 라덴 bioaerosols 생성할 수 설명 바람 속도, 고 졸 대량 농도입니다. 이 프로토콜을 사용 하 여의 주요 장점은 수정 되지 않은 기존의 액체 임 핀 저를 직접 비교를 통해 평가 바이러스 탐지의 감도 증가입니다. 다른 장점은 집중 다양 한 부정 청구 바이러스, 음이온 교환 수 지 (샘플 당 ~$0.14), 및 사용의 용이성의 저렴 한 비용을 포함 합니다. 단점 수 지 흡착 바이러스 성 입자의 infectivity 평가이 프로토콜의 무 능력을 포함 하 고 액체 샘플링 버퍼의 최적화에 대 한 필요는 임 핀 저 내에서 사용 하는 잠재적으로.

그림 1 에서 bioaerosols 액체 기반의 impingers 수 지의 포함을 통해 바이러스의 차지 기반 캡처의 뒤에 원리를 보여 줍니다. 그림 2 는 사용자 bioaerosol 챔버의 설치. 그림 3 에서는 aerosolization 실험 및 측정 품질 관리를 보장 하기 위해 설정에 관련 된 단계를 설명 합니다. 그림 4 는 qRT-PCR bio...

Watch this Scientific Journal Video about Detection of Viruses from Bioaerosols Using Anion Exchange Resin at JoVE.com

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

음이온 교환 수 지 기반 방법, 바이러스의 충돌-기반 bioaerosol 샘플링 시연 액체에 적응 합니다. 다운스트림 분자 탐지와 결합 하면 메서드 bioaerosols에서 바이러스의 손쉬운 및 민감한 감지.

Bioaerosols 음이온 교환 수 지를 사용 하 여 바이러스의 검출

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid ...

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8.1K 조회수.  Colorado State University. 음이온 교환 수 지 기반 방법, 바이러스의 충돌-기반 bioaerosol 샘플링 시연 액체에 적응 합니다. 다운스트림 분자 탐지와 결합 하면 메서드 bioaerosols에서 바이러스의 손쉬운 및 민감한 감지.

비디오: Detection of Viruses from Bioaerosols Using Anion Exchange Resin

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist&#160;of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (&#181;PADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the &#181;PAD. Alternatively, digital images of the reacted &#181;PADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24&#160;hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (&#181;PAD) imbedded with bacterial-indicative colorimetric substrates.

색도계 탐지의 종이 기반의<em&gt; 대장균</em&gt;<em&gt; 살모넬라</em&gt; SPP., 그리고<em&gt; 리스테리아</em&gt; 농업 다량의 물에서

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of ...

연구

환경과학

Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the biogeochemical drivers of climate change and to the development and evaluation of GHG mitigation strategies based on modulation of landscape management practices. The static chamber-based method described here is based on trapping gases emitted from the soil surface within a chamber and collecting samples from the chamber headspace at regular intervals for analysis by gas chromatography. Change in gas concentration over time is used to calculate flux. This method can be utilized to measure landscape-based flux of carbon dioxide, nitrous oxide, and methane, and to estimate differences between treatments or explore system dynamics over seasons or years. Infrastructure requirements are modest, but a comprehensive experimental design is essential. This method is easily deployed in the field, conforms to established guidelines, and produces data suitable to large-scale GHG emissions studies.

This article showcases the static chamber-based method for measurement of greenhouse gas flux from soil systems. With relatively modest infrastructure investments, measurements may be obtained from multiple treatments/locations and over timeframes ranging from hours to years.

정적 챔버를 사용하여 농업 토양에서 온실 가스 유출 측정

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the ...

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.

A PCR-based protocol was adapted to detect Cronobacter spp., Salmonella enterica, and Listeria monocytogenes from body surfaces and alimentary canals of individual wild-caught flies. The goal of this protocol is to detect and isolate bacterial pathogens from individual insects collected as part of an environmental sampling program during foodborne outbreak investigations.

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a ...

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography, Deans Switching and Sulfur-selective Detection

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate interferences, a two dimensional column configuration is used, with a Deans switch employed to transfer hydrogen sulfide from the first to the second column (heart-cutting). Liquid crude samples are first separated on a dimethylpolysiloxane column, and light gases are heart-cut and further separated on a bonded porous layer open tubular (PLOT) column that is able to separate hydrogen sulfide from other light sulfur species. Hydrogen sulfide is then detected with a sulfur chemiluminescence detector, adding an additional layer of selectivity. Following separation and detection of hydrogen sulfide, the system is backflushed to remove the high-boiling hydrocarbons present in the crude samples and to preserve chromatographic integrity. Dissolved hydrogen sulfide has been quantified in liquid samples from 1.1 to 500 ppm, demonstrating wide applicability to a range of samples. The method has also been successfully applied for the analysis of gas samples from crude oil headspace and process gas bags, with measurement from 0.7 to 9,700 ppm hydrogen sulfide.

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography, Deans Switching and Sulfur-selective Detection

A multidimensional gas chromatography method for the analysis of dissolved hydrogen sulfide in liquid crude oil samples is presented. A Deans switch is used to heart-cut light sulfur gases for separation on a secondary column and detection on a sulfur chemiluminescence detector.

H의 측정<sub&gt; 2</sub&gt; 원유 및 다차원 가스 크로마토 그래피를 이용하여 원유 헤드 스페이스에서 S는 학장은 전환과 황 선택적 검출

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate ...

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in nutrient and gas cycles. After cores with intact sediment-water interfaces are collected, they are submerged in incubation tanks and kept under aerobic conditions at in situ temperatures. To initiate a time course of overlying water chemistry, cores are sealed without bubbles using a top cap with a suspended stirrer. Time courses of 4-7 sample points are used to determine the rate of sediment water exchange. Artificial illumination simulates day-time conditions for shallow photosynthetic sediments, and in conjunction with dark incubations can provide net exchanges on a daily basis. The net measurement of N2 is made possible by sampling a time course of dissolved gas concentrations, with high precision mass spectrometric analysis of N2:Ar ratios providing a means to measure N2 concentrations. We have successfully applied this approach to lakes, reservoirs, estuaries, wetlands and storm water ponds, and with care, this approach provides valuable information on biogeochemical balances in aquatic ecosystems.

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

Here, we present small core incubations for the measurement of sediment-water gas and solute exchange. These will provide reliable measurements of sediment-water exchange that assess the role of sediment in influencing biological and biogeochemical processes in aquatic ecosystems.

O의 저서 교환<sub&gt; 2</sub&gt;, N<sub&gt; 2</sub작은 코어 항온 사용&gt;와 용존 영양소

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in ...

Building Double-decker Traps for Early Detection of Emerald Ash Borer

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions of forest and landscape ash (Fraxinus spp.) trees. Several artificial trap designs to attract and capture EAB beetles have been developed to detect, delineate, and monitor infestations. Double-decker (DD) traps consist of two corrugated plastic prisms, one green and one purple, attached to a 3 m tall polyvinyl chloride (PVC) pipe supported by a t-post. The green prism at the top of the PVC pipe is baited with cis-3-hexenol, a compound produced by ash foliage. Surfaces of both prisms are coated with sticky insect glue to capture adult EAB beetles. Double-decker traps should be placed near ash trees but in open areas, exposed to sun. Double-decker trap construction and placement are presented here, along with a summary of field experiments demonstrating the efficacy of DD traps in capturing EAB beetles. In a recent study in sites with relatively low EAB densities, double-decker traps captured significantly more EAB than green or purple prism traps or green funnel traps, all of which are designed to be suspended from a branch in the canopy of ash trees. A greater percentage of double decker traps were positive, i.e., captured at least one EAB, than the prism traps or funnel traps that were hung in ash tree canopies.

Effective traps to attract and capture the emerald ash borer (EAB) are a key element of detecting and managing this invasive pest. Double-decker traps, placed in full sun near ash trees, incorporate visual and olfactory cues and were more likely to capture EAB than other trap designs in field trials.

에메랄드 색 재 송곳의 조기 발견을 위한 이층 트랩을 구축

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions ...

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

Rapid and accurate detection of plant pathogens on-site, especially soil-borne pathogens, is crucial to prevent further inoculum production and proliferation of plant diseases in the field. The method developed here using a portable real-time PCR detection system enables on-site diagnosis under field conditions.

휴대용 실시간 PCR 시스템을 사용 하 여 토양 품 어진 Phytopathogens의 현장 분자 검출

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management ...

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf mottling, vein yellowing, defoliation, root decay, and ultimately, the death of the citrus plant. When infected, the citrus plants have stunted growth and produce flowers out of season. These flowers rarely yield fruit, and those that do yield small, bitter, irregularly shaped citrus fruit that are not desirable. This disease is spread by the Asian citrus psyllid, Diaphorina citri, and by the grafting of infected citrus tissue. The pathogen has a long and variable incubation period within the citrus plant&#8212;sometimes years, before symptoms appear. Attempts to culture this pathogen in vitro have been unsuccessful, possibly due to the low and uneven concentration of the pathogen within infected citrus tissue, or because it is difficult to replicate the environmental conditions conducive to growth of the pathogen. It is very difficult to identify the disease before it has spread, due to its long incubation period and researchers&#39; inability to culture the pathogen. As a result, the disease only becomes apparent after suddenly destroying a citrus farmer&#39;s entire yield. Presented here is a method for the accurate and specific detection of the citrus greening pathogen, Candidatus liberibacter spp. using a genomic DNA extraction kit and PCR. This method is simple, efficient, cost effective, and adaptable for quantitative analysis. This method can be adapted for use on any citrus tissue; however, it is potentially limited by the amount of pathogen present in the tissue. Nevertheless, this method will allow citrus farmers to identify infected citrus plants earlier, and curb the spread of this destructive disease before it can further spread.

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter  spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Citrus Greening is a particularly destructive disease affecting citrus crops globally. Presented here is a simple method using PCR and genomic DNA extraction of citrus leaf tissue for the accurate and precise identification of the citrus greening pathogen, Candidatus liberibacter spp.

특정 및 감귤 류 녹화 병원 체 Candidatus liberibacter 종 감귤 잎 조직 샘플에서 사용 하 여 기존의 PCR의 정확한 검출

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf ...

An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to bacteria control the amount that will accumulate in staple food crops. The bioavailability of these metals is dependent on their speciation in solution, but more particularly under anoxic conditions where Hg is methylated to toxic monomethylmercury (MeHg) and Cd persists in the rhizosphere. Whole-cell microbial biosensors give a quantifiable signal when a metal enters the cytosol and therefore are useful tools to assess metal bioavailability. Unfortunately, most biosensing efforts have so far been constrained to oxic environments due to the limited ability of existing reporter proteins to function in the absence of oxygen. In this study, we present our effort to develop and optimize a whole-cell biosensor assay capable of functioning anaerobically that can detect metals under anoxic condition in quasi-real time and within hours. We describe how the biosensor can help assess how chemical variables relevant to the environmental cycling of metals affect their bioavailability. The following protocol includes methods to (1) prepare Hg and Cd standards under anoxic conditions, (2) prepare the biosensor in the absence of oxygen, (3) design and execute an experiment to determine how a series of variable affects Hg or Cd bioavailability, and (4) to quantify and interpret biosensor data. We show the linear ranges of the biosensors and provide examples showing the method's ability to distinguish between metal bioavailability and toxicity by utilizing both metal-inducible and constitutive strains.

Here, we present a protocol to use an anaerobic whole-cell microbial biosensor to evaluate how different environmental variables affect the bioavailability of Hg and Cd to bacteria in anoxic environments.

수은 카드뮴의 검출에 대 한 혐 기성 바이오 센서 분석 결과

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to ...

A Precise and Autonomous System for the Detection of Insect Emergence Patterns

Existing systems to measure insect emergence patterns have limitations; they are only partially automated and are limited in the maximum number of emerging insects they can detect. In order to obtain precise measurement of insect emergence, it is necessary for systems to be semi-automated and able to measure large numbers of emerging insects. We addressed these issues by designing and building a system that is automated and can measure emergence of up to 1200 insects. We modified the existing &#34;falling-ball&#34; system using Arduino microcontrollers to automate data collection and expand the sample size through multiple data channels. Multiple data channels enable the user to not only increase their sample size, but also allows for multiple treatments to be run simultaneously in a single experiment. Furthermore, we created an R script to automatically visualize the data as a bubble plot, while also calculating the median day and time of emergence. The current system was designed using 3D printing so the user can modify the system to be adjusted for different species of insects. The goal of this protocol is to investigate important questions in chronobiology and stress physiology, using this precise and automated system to measure insect emergence patterns.

Measurement of insect emergence patterns requires precision. Existing systems are only semi-automated and sample size is limited. We addressed these issues by designing a system using microcontrollers to precisely measure the time of emergence of large numbers of emerging insects.