We would like to thank Dr. Nicholas J. Ashbolt and Dr. G. Shay Fout for their review of the manuscript.

유리 및 필터 하우징 1. 준비 <ol style="margin-left: 40px;"><li> 특별히 언급하지 않는 한, 15 분 동안 121 ° C에서 모든 유리, 필터 하우징 및 솔루션을 소독. 불임을 보장하기 위해 모든 개구 또는 알루미늄 호일 또는 이전 살균에 테이프 확보 종이 중 하나에 노출 된 표면을 커버합니다. </li><li> 1 L 사이드 암 삼각 플라스크에 필터 하우징 (47mm 직경)를 부착하여 여과 장치를 조립합니다. 필요한 필터를 수집 : 47mm 직경 전기 음성 / 전기 양성 디스크 필터를. </li><li> 1 L의 1.5 %의 쇠고기 추출물 준비 (응집을 플록 입자를 생성 용액의 pH를 3.5, 또는 비 응집 저하되면, pH를 3.5에서 골재를하지 않는) 비프 엑기스 15g에 용해시켜, 0.05 M 글리신, 1 탈 이온수의 L과 3.75 g 글리신을 추가. 주 : 비 응집 쇠고기 추출물 전에 급 농도 셀 라이트의 첨가를 필요로 할 것이다. </li><li> 용출 첨가제 나트륨 polyph 추가0.1 % 농도에서 쇠고기 추출물 osphate. </li><li> 1 M 염산 용액을 첨가 쇠고기 추출물, 원하는 산도로 pH를 혼합 용액에 적가 하였다. 121 ℃에서 15 ~ 30 분간 오토 클레이브 쇠고기 추출물. </li><li> 잘, 매체, 또는 (비 응집 쇠고기 추출물 만 함께 사용) 큰 입자 셀 라이트 0.1 g을 측정합니다. </li><li> 삼각 부착 플라스크에 진공 출구에 대응 튜브를 부착함으로써 진공 / 흡착을 준비한다. </li></ol> 솔루션 및 바이러스 재고 2. 준비 <ol style="margin-left: 40px;"><li> 1 M HCl을 1 L를 준비합니다. </li><li> 1 M NaOH를 1 L를 준비합니다. </li><li> 1X PBS 용액을 준비합니다 (137 mM의 NaCl을, 2.7 mM의 KCl을, 1.47 mM의 K 2 PO 4, 4.3 mM의의 NaH 2 PO 4). <ol><li> 1 M의 NaOH를 사용하여 9.0 1X PBS의 산도를 조정합니다. </li></ol></li><li> 준비하고 9 ㎖의 PBS (PH와 재고 바이러스 1 ㎖를 혼합하여 10 4 -10 5 가장 가능성있는 수 (MPN) ml의 -1 스톡 바이러스를 희석7), 8, 13을 앞서 기재된. 1 ml의 분취 량에서 -80 ° C에서 보관. <ol><li> 잠재적 오염물을 제거하기 위해 필터링 주사기 (0.22 ㎛의 공극 크기)에 의해 바이러스 스톡 멸균. </li></ol></li><li> 탈 염소 수돗물 <ol><li> 측정 값을 이용하여 무균 살균 수돗물 1 L 눈금 실린더 및 자기 교반 막대를 함유하는 2 L 비이커에 붓는다. </li><li> 티오 황산나트륨 0.7 g을 추가하고 입자가 용해 될 때까지 섞는다. 1 M 염산 또는 1 M NaOH 용액을 사용하여 필요한 경우 7-7.5에 수돗물의 산도를 조정합니다. </li><li> 해동 바이러스 스파이크 (1 mL) 및 탈 염소 살균 수돗물 1 L로 스파이크의 전체 볼륨. 10 분 동안 혼합한다. </li><li> 스파이크 된 샘플과 동일한 방법으로 각각의 실험 과정에있어서 블랭크 (아데노 않고 탈 염소 수돗물)을 준비한다. </li></ol></li></ol> 밀리 포어 필터 장치의 제조 3 <ol style="margin-left: 40px;"><li> 필터 하우징을 통해 멸균 덮개를 제거합니다. 확인필터 하우징 상부 그릇과 낮은 필터 스크린 사이의 적절한 맞춤 여과시 시료 누출을 방지 할 수 있습니다. </li><li> 1 L 삼각 플라스크에서 멸균 덮개를 제거합니다. 좋은 도장을 만들어, 필터 유닛의 코르크 삼각 플라스크의 상단 구멍에 꼭 맞는지 확인합니다. </li><li> 낮은 필터 화면에서 필터 유닛 위에 그릇을 제거하고 멸균 집게로 화면을 통해 정면으로 47mm 전기 양성 필터 디스크를 넣습니다. 화면 하단에 필터 하우징 그릇을 부착하고 시계 반대 방향으로 비틀 어서 제자리에 고정. </li></ol> 4. 물 여과 (기본 농도) <ol style="margin-left: 40px;"><li> 바이러스 100 ml의 멸균 졸업 된 실린더 사용하여 수돗물 아군 측정한다. </li><li> 바이러스 100ml를 47mm 플리츠 유리 / 셀룰로오스 필터 또는 나노 알루미나 / 유리 섬유 디스크 필터를 포함하는 필터 하우징 내로 수돗물 아군 붓는다. </li><li> 물이 천천히 필터를 통과하도록 허용합니다. 오 남아있는 잔여 물을 제거하기 위해 부드러운 진공 적용필터 표면에서 F 수돗물. </li><li> 트립신 처리를 사용하는 경우, 표면을 필터링하고 5 분 동안 배양 0.25 % 트립신 5 ㎖를 추가합니다. </li><li> 멸균 탈 50 ml의 물을 사용하여 필터 표면에서 트립신을 씻으십시오. </li></ol> 필터 5. 바이러스 용출 <ol style="margin-left: 40px;"><li> 2 L 삼각 플라스크에서 필터 하우징을 제거하고, 250 ml의 사이드 암 플라스크에 배치합니다. </li><li> 눈금 실린더를 사용 하 쇠고기 추출물 100 ㎖를 측정한다. 천천히 필터 하우징에 쇠고기 추출물 100 ㎖를 붓는다. </li><li> 쇠고기 추출물은 작은 (250 ml)을 삼각 플라스크에 쇠고기 추출물을 캡처, 필터를 통해 붓는다. 필터에서 잔류 쇠고기 추출물을 뽑아 진공을 적용합니다. </li></ol> 6. 셀 라이트 보조 농도 <ol style="margin-left: 40px;"><li> 볶음 판에 용출 된 바이러스를 포함하는 전송 플라스크. 약간의 소용돌이를 만들 혼합, 교반 접시에 무균 자기 교반 막대와 장소를 추가합니다. </li><li> 셀 라이트 0.1 g을 넣고쇠고기 추출물 100 ㎖에 분말과 셀 라이트를 분산 할 수 있습니다. 쇠고기 추출물로 멸균 산도 프로브를 놓습니다. </li><li> pH가 4.0을 달성하기 위해 쇠고기 추출물에 현명한 1 M 염산을 떨어 추가합니다. 천천히 10 분 동안 혼합 할 수 있습니다. </li><li> 2 L 삼각 플라스크를 사용하여 (전술 한 바와 같이) 필터 하우징 상에 47mm 프리 필터를 놓습니다. </li><li> 프리 필터를 통해 100 ml의 쇠고기 추출물 / 셀 라이트 혼합물을 붓고. 쇠고기 추출물을 통해 붓는다. 필터에서 잔류 쇠고기 추출물을 뽑아 약간의 진공을 적용합니다. </li><li> 필터 하우징 배출구의 끝에 금속 튜브 클립을 놓습니다. 금속 튜브 클립에 멸균 15 ml의 폴리 프로필렌 수집 튜브를 연결합니다. 다시 삼각 플라스크에 넣고 필터 하우징을 놓습니다. </li><li> 프리 필터에 포집 셀 라이트를 통해 1X PBS 버퍼 5 ㎖, pH가 9.0를 추가합니다. </li><li> PBS는 프리 필터를 통해 물방울을 허용합니다. 15 ml의 수집 관에 잔류 PBS를 통해 당기하려면 약간의 진공을 적용합니다. </li></ol> 7. 유기 응집 보조 농도 <ol style="margin-left: 40px;"><li>볶음 판에 용출 된 바이러스가 들어있는 비커를 전송합니다. 쇠고기 추출물을 100 ml의 멸균 자기 교반 막대를 추가하고 약간의 소용돌이를 만들 섞는다. </li><li> 쇠고기 추출물로 멸균 산도 프로브를 놓습니다. </li><li> 쇠고기 추출물 현명한 1 M 염산 드롭을 추가하고 3.5로 산도를 조정합니다. 30 분 동안 혼합한다. </li><li> 4 ºC에서 15 분 동안 2,500 XG에 250 ml의 원심 분리기 원뿔 튜브에 용출액을 원심 분리기를 따르십시오. </li><li> 펠렛을 방해하지 않도록 상층 액을 복용 치료를 붓고. 1X PBS, pH가 9 5ml에 펠렛을 재현 탁. </li><li> 4 ºC에서 10 분 동안 4,000 XG에 현탁액을 원심 분리기. 상층 액을 붓고 펠렛을 폐기합니다. </li><li> 7-7.5로 pH를 조정, 필터는 -80 ° C에서 0.22 μm의 주사기 필터 및 동결을 통해 소독. </li></ol> 8. 바이러스 핵산 추출 <ol style="margin-left: 40px;"><li> 제조업체의 지침에 따라 적절한 상용 키트를 사용하여 바이러스 핵산을 추출합니다. </li></ol> &lt;P 클래스 = &quot;jove_title&quot;&gt; 9. ADV 40/41 qPCR에 <ol style="margin-left: 40px;"><li> 8, 13을 발표 한 프라이머 및 프로브뿐만 아니라, 반응 혼합물과 열 순환 프로파일을 사용합니다. 주 : 분석의 검출의 예상 한계 반응 당 약 5 qPCR의 단위입니다. </li><li> 다음과 같은 농도에서 PCR 증폭 혼합물의 구성 요소를 추가 : 10 mM 트리스 (PH 8.3)의 50 mM의 KCl을, 4.5 mM의의 MgCl 2, 10 mM의 dNTPs를, 10 μM 프라이머 1 μm의 프로브와 중합 효소 0.5 μL 그래서 45의 최종 볼륨 μL를 96- 웰 PCR 반응 플레이트 내에 웰의 적절한 수에 첨가 될 수있다. </li><li> 96 웰, 빠른 반응 PCR 플레이트의 각 해당 잘으로 PCR 증폭 혼합물의 부하 45 μL. </li><li> DNA의 5 μL는 PCR 플레이트의 적절한 우물에 샘플을 추출 추가합니다. </li><li> , 내열성 커버 씰 플레이트를 커버 PCR 판 우물의 바닥에있는 방울을 이동 판을 회전. </li><li> foll으로 설정 PCR 증폭주기OWS : 15 초 동안 95 ° C의 40주기와 1 분 60 ° C 다음에 10 분 동안 95 ° C. </li></ol> 10. 데이터 분석 <ol style="margin-left: 40px;"><li> 5에서 1 : 1의 샘플에 걸친 일련의 5 배 희석액 중으로 측정 분석 (625)의 희석 범위를 바이러스 입자의 가장 가능한 번호 (MPN)을 결정하기 위해, 8, 13을 앞서 설명 된 바와 같이. </li><li> 5에서 1 : 1 아데노 바이러스 실험을 걸친 일련의 스파이크 5 배 희석액을 분석 15,625 희석 범위를 바이러스 입자의 가장 가능한 번호 (MPN)을 결정하기 위해. </li><li> EPA MPN 계산기 (http://www.epa.gov/nerlcwww/online.html)를 사용하여, 희석의 유형을 고려하여 조정 계산기 (1 : 5 1:10), 희석 수 행하고, 숫자 일련의 희석에 샘플을 복제. </li><li> 수행 볼륨 단위로 등 (예를 들면, 1 ㎖, 100 ㎖를 정규화하여 다음 데이터의 열 변형 로그 </EM&gt;). 샘플에 존재하는 바이러스 입자의 수​​를 나누어 주어 실험에 대한 회수율을 계산 실험 스파이크에 존재하는 바이러스 입자의 수​​에 집중하고 있습니다. </li><li> 다른 실험 변수의 효과를 평가 (예를 들어, 다른 셀 라이트 타입의 pH 범위에서, 다른 필터 타입과 상이한 이차 농도 기법) (예를 들면, 짝 t 검정, 하나 또는 두 개의 파라 메트릭 또는 비 - 파라 메트릭 통계 테스트를 수행하여 ADV 회수율에 데이터의 분배 및 실험 디자인에 따라)을 - 방향 ANOVA. </li></ol>

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L., 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=Round+robin+investigation+of+methods+for+the+recovery+of+poliovirus+from+drinking+water.">Round robin investigation of methods for the recovery of poliovirus from drinking water.</a> Applied and Environmental Microbiology. 47, 144-150 (1984).</li><li>Schwab, K. J., De Leon, R., Sobsey, M. 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The views expressed in this article are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

전기 양성 필터는 물에서 바이러스를 집중에 유용하다; 그러나 이러한 필터는 다시 자신의 효과를 변경할 수있는 그들의 구조와 구성이 다를 수 있습니다. 이 문제를 복잡하게, 캡시드 구조와 요금 농도 기술은 최적의 복구 (15)을 보장하기 위해 맞출 필요 바이러스 균주에 따라서 다릅니다. (예를 들어, 전기 양성 필터, 쇠고기 추출물 용출), 대상 바이러스의보다 효과적인 농도 ...

인간의 장내 바이러스는 수 인성 질병에 1-3의 중요한 원인이 요원하지만, 농도없이 자신의 검출이 어려워 일반적으로 오염 된 환경 바다에서 낮은 숫자에 존재한다. 바이러스를 농축하는데 사용 된 절차는 일반적으로 필터 용출액 여과 필터 뒤에 용출 단계, 및 보조 농도를 포함한다. 일반적인 여과 절차 (최근 4,5 검토) 등 전기 양성 필터와 같은 충전 세포막의 사용에 의존한다. 이 필터는 필터 표면 (양전하)과 대상 바이러스 입자 사이의 정전 기적 상호 작용을 사용하여 물에 현탁 바이러스를 캡처에 의존 (음전하). 시판되는 두 전성 필터가이 기술에 의존하는, 유리 / 셀룰로오스 나노 알루미나 / 유리 섬유 필터. 유리 / 셀룰로오스 필터 비용이 유리 (/ CE)의 사용을 제한하는 최대 10 배 나노 알루미나 / 유리 섬유의, 아르일상 바이러스 모니터링을위한 llulose 필터. 최근 연구에 싼 필터 대안의 사용을 정당화 차이가 주변 물 6,7에서 엔테로 바이러스의 회복이 두 필터 사이의 공칭 결론을 내렸다. 이러한 전성 유리 양모 필터와 같은 다른 필터 옵션 그러나, 그들은 하나의 소스 물 (전기 음성 필터)의 전처리가 필요하거나 (유리 양모 필터) 상업적으로 사용할 수 없습니다, 연구되고있다. 바이러스 농도 절차의 개발은 주로 물에서 바이러스 회수율을 향상시키기 위하여 기본 농도 기법 (필터)의 최적화에 초점을 맞추고있다. 그러나 볼륨 밀리리터 전형적으로 1 L로부터 얻은 용출액 부피를 감소 이차 농도 절차는 또한 바이러스 회수율 8에 상당한 영향을 미칠 수있다. 장용성 바이러스의 농도는 일반적으로 보조 소고기 추출물 일부 유형으로 응집제에 의존 (유기 floccu만큼은) 또는 무 지방 우유 응집 9-12 필터 표면에서 바이러스 입자를 제거합니다. 최근, 셀 라이트 (미세 입자)의 첨가와 결합 쇠고기 추출물을 사용하여 다른 농도 차 절차는 아데노 바이러스, 장내 바이러스, 및 노로 8,13,14의 회수 가능성을 보여 주었다. 이 바이러스 입자에 유기 응집 방법과 유사한 원리 하에서 셀 라이트 농도 작품에 부착 및 현탁액의 pH를 변경하여 입자 (플록 또는 셀 라이트)로부터 방출된다. 이 두 차의 농도 기술 사이의 비교가 아군 아데노 바이러스 (ADV) 유형 40, 41 (8)의 회복으로 평가되고있다.이 연구는 두 개의 보조 농도 기술은 아데노 바이러스의 복구에 통계적으로 차이가 없었다 결론을 내렸다. 그러나, 유기 응집 방법은 30 분을 필요로한다. pH가 3.5에서 배양, 셀 라이트 기술은 pH가 4.0에서 짧은 배양 (10 분)을 필요로한다. 유기 flocculat이온도 급 농도 중에 플록 입자를 수집 비싼 실험 장비 (원심)의 사용을 필요로, 반대로 셀 라이트 기술은 현탁액으로부터 셀 라이트 입자를 분리하는 기본적인 실험 장비 (진공 여과)를 사용한다. 필터와 이차 용출 기술 특정 조합은 또한 바이러스 회수율에 영향을 미칠 수있다. 한 연구는 주 (전기 양성 필터)의 특정 조합 및 보조 농도 기술 (셀 라이트 또는 유기 응집이) 아데노 바이러스 (13)의 회복에 상당한 영향을 미쳤다 결론을 내렸다. 이러한 연구 결과는 이러한 기술을 사용할 때 그 최적화가 최적으로 주어진 물 행렬로부터 대상 바이러스를 회수하기 위해 필요한 제안한다. 최적화는 여러 변수 (필터 유형 / 브랜드, 산도 용출 용액, 셀 라이트 / 유기 응집을) 평가되기 때문에, 힘든 과정을 많은 연구자들이 적극적으로 방지 걸리는 시간입니다. 이 초간각 바이러스 유형 고유 캡시드 형태 및 특정 캡시드 전하 보여주는데 tudy이 절차는 아마도 아군 인간 아데노 바이러스를 40 균주 41 이용한 물에서 바이러스 농도에 대한 최적의 조건을 식별하기 위해 개발되었다 농도 프로토콜은 모든 바이러스에 대해 최적화 될 필요가있다 최적 바이러스 복구를 달성하기 위하여 대상. 1) 전기 양성 필터 2 다음에 디스크) 보조 농도 셀 라이트 기술에 비해 설립 유기 응집 방법의 평가를 사용하여 수돗물의 바이러스 복구를 평가, 그리고 3) 평가 :이 연구는 ADV (40)에 의해 41 농도에 대한 접근 방법을 제공합니다 차 농도에 대한 용출 버퍼.

<table><tbody><tr><td>Adenovirus 40 stock</td><td>ATCC</td><td>VR-931</td><td></td></tr><tr><td>Adenovirus 41 stock</td><td>ATCC</td><td>VR-930</td><td></td></tr><tr><td>Sodium Thiosulfate</td><td>Fluka Chemical Co.</td><td>72051</td><td></td></tr><tr><td>Celites #577</td><td>Fluka Chemical Co.</td><td>22142</td><td></td></tr><tr><td>NanoCeram 47 mm</td><td>Argonide</td><td>N/A</td><td></td></tr><tr><td>1MDS 47 mm</td><td>3M</td><td>6408502</td><td></td></tr><tr><td>AP-20 Prefilter 47 mm</td><td>Millipore Corp.</td><td>AP2004700</td><td></td></tr><tr><td>Glycine&amp;nbsp;</td><td>Sigma</td><td>50046-1KG</td><td></td></tr><tr><td>Sodium Polyphosphate</td><td>Acros Organics</td><td>390930010</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25200</td><td></td></tr><tr><td>PBS</td><td>Sigma</td><td>P5368</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Fisher</td><td>A481-212</td><td></td></tr><tr><td>BBL Beef Extract</td><td>BD Biosciences</td><td>212303</td><td></td></tr><tr><td>Difco Beef Extract</td><td>BD Biosciences</td><td>211520</td><td></td></tr><tr><td>ABI 7900 Real-time PCR system</td><td>ABI</td><td>N/A</td><td></td></tr><tr><td>Stainless Steel Filter Housing</td><td>Millipore Corp.</td><td>XX2004720</td><td></td></tr><tr><td>Blood DNA Extraction Kit</td><td>Qiagen&amp;nbsp;</td><td>51104</td><td></td></tr><tr><td>EPA MPN Calculator</td><td></td><td></td><td>http://www.epa.gov/nerlcwww/online.html</td></tr></tbody></table>

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.

<html> 셀 라이트 선택 셀 라이트의 세 가지 다른 종류의 이전 실적이 가장 좋은 변종의 선택을 시험 하였다. 중간 크기의 입자를 미세과 Celites 가장 높은 아데노 바이러스 복구를 생산했다. 큰 celites의 사용은 AdV40 41 (범위 32 % -100 %) (그림 1) 모두 회수율 결과. 아데노 바이러스 (40)의 평균 회수율은 1백44퍼센트 ± 52 % (미세), 115 % ± 28 % (중간) 및 82 % ± 53 % (대형) 입자 ...

Watch this Scientific Journal Video about A Small Volume Procedure for Viral Concentration from Water at JoVE.com

A Small Volume Procedure for Viral Concentration from Water

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 ...

a-small-volume-procedure-for-viral-concentration-from-water

Universidad San Sebastian

Research

JoVE Journal

Environment

10.0K 조회수.  U.S. EPA. 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. 

비디오: A Small Volume Procedure for Viral Concentration from Water

Estimating Virus Production Rates in Aquatic Systems

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative estimates of this process is critical as we can then develop better models of microbial community structure and function as well as advance our understanding of how viruses work to alter aquatic biogeochemical cycles. The virus reduction technique allows researchers to estimate the rate at which virus particles are released from the endemic microbial community. In brief, the abundance of free (extracellular) viruses is reduced in a sample while the microbial community is maintained at near ambient concentration. The microbial community is then incubated in the absence of free viruses and the rate at which viruses reoccur in the sample (through the lysis of already infected members of the community) can be quantified by epifluorescence microscopy or, in the case of specific viruses, quantitative PCR. These rates can then be used to estimate the rate of microbial mortality due to virus-mediated cell lysis.

The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.

아쿼틱 시스템에서 바이러스 생산 요금 예측

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

연구

면역학 및 감염병학

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

물 화학 및 해양 미생물에 필드 가이드 - 바다에서 보이지 않는 선수를 해명

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

환경과학

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency&#8217;s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.

Here we present a procedure to measure total culturable viruses using the Buffalo Green Monkey kidney cell line. The procedure provides a standardized tool for measuring the occurrence of infectious viruses in environmental and drinking waters.

엔테로 바이러스 및 문화 RT-qPCR에 의해 물에서 노로 바이러스의 발생의 EPA 방법 1615 측정. II. 총 배양 가능한 바이러스 분석

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. ...

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 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

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 ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

특정 인간과 동물 바이러스를 사용하여 미생물 소스 추적을위한 비용 효율적인 방법

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Nucleocapsids에서 바이러스성 DNA의 준비

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens

The key first step in evaluating pathogen levels in suspected contaminated water is concentration. Concentration methods tend to be specific for a particular pathogen group, for example US Environmental Protection Agency Method 1623 for Giardia and Cryptosporidium1, which means multiple methods are required if the sampling program is targeting more than one pathogen group. Another drawback of current methods is the equipment can be complicated and expensive, for example the VIRADEL method with the 1MDS cartridge filter for concentrating viruses2. In this article we describe how to construct glass wool filters for concentrating waterborne pathogens. After filter elution, the concentrate is amenable to a second concentration step, such as centrifugation, followed by pathogen detection and enumeration by cultural or molecular methods. The filters have several advantages. Construction is easy and the filters can be built to any size for meeting specific sampling requirements. The filter parts are inexpensive, making it possible to collect a large number of samples without severely impacting a project budget. Large sample volumes (100s to 1,000s L) can be concentrated depending on the rate of clogging from sample turbidity. The filters are highly portable and with minimal equipment, such as a pump and flow meter, they can be implemented in the field for sampling finished drinking water, surface water, groundwater, and agricultural runoff. Lastly, glass wool filtration is effective for concentrating a variety of pathogen types so only one method is necessary. Here we report on filter effectiveness in concentrating waterborne human enterovirus, Salmonella enterica, Cryptosporidium parvum, and avian influenza virus.

Glass wool filters have been used to concentrate waterborne viruses by a number of research groups around the world. Here we show a simple approach for constructing glass wool filters and demonstrate the filters are also effective in concentrating waterborne viral, bacterial and protozoan pathogens.

집중 Waterborne 바이러스와 농업 Zoonotic 병원균에 대한 유리 양모 필터

The key first step in evaluating pathogen levels in suspected contaminated water is concentration.  Concentration methods tend to be specific for a ...

Concentration of Virus Particles from Environmental Water and Wastewater Samples Using Skimmed Milk Flocculation and Ultrafiltration

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities.&#160;The recovery of microbial fractions, including viruses, bacteria, and microeukaryotes from wastewater and environmental water samples&#160;is one of the challenging steps in these approaches. In this study, we focused on the recovery efficiency of sequential ultrafiltration and skimmed milk flocculation (SMF) methods using Armored RNA as a test virus, which is also used as a control by some other studies. Prefiltration with 0.45 &#181;m and 0.2 &#181;m membrane disc filters were applied to eliminate solid particles before ultrafiltration to prevent the clogging of ultrafiltration devices. Test samples, processed with the sequential ultrafiltration method, were centrifuged at two different speeds. An increased speed resulted in lower recovery and positivity rates of Armored RNA.&#160;On the other hand, SMF resulted in relatively consistent recovery and positivity rates of Armored RNA. Additional tests conducted with environmental water samples demonstrated the utility of SMF to concentrate other microbial fractions. The partitioning of viruses into solid particles might have an impact on the overall recovery rates, considering the prefiltration step applied before the ultrafiltration of wastewater samples. SMF with prefiltration performed better when applied to environmental water samples&#160;due to lower solid concentrations in the samples and thus lower partitioning rates to solids. In the present study, the idea of using a sequential ultrafiltration method arose from the necessity to decrease the final volume of the viral concentrates during the COVID-19 pandemic, when the supply of the commonly used ultrafiltration devices was limited, and there was a need for the development of alternative viral concentration methods.

Virus concentration from environmental water and wastewater samples is a challenging task, carried out primarily for the identification and quantification of viruses. While several virus concentration methods have been developed and tested, we demonstrate here the effectiveness of ultrafiltration and skimmed milk flocculation for RNA viruses with different sample types.

탈지유 응집 및 한외여과를 사용한 환경 용수 및 폐수 샘플의 바이러스 입자 농도

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities.&#160;The ...

Lentivirus Production

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

Lentivirus 제작

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

생물학

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

사용 환경 샘플에서 새로운 Faustoviruses의 분리에 대한 신속한 전략<em&gt; Vermamoeba의 vermiformis</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...