우리는 비디오를 해설을 위해 윌리엄 T. Eckert 감사드립니다. 글라스 울 프로토콜의 개발은 미국 EPA는 스타 그랜트 R831630로부터 자금 장용 위험 (WAHTER 연구)에 대한 위스콘신 물과 건강 평가판의 일부였다. 알래스카 샘플은 US 지질로부터 재정 지원으로 A. 리브스, A. Ramey,와 B Meixell이 수집되었다. 무역, 제품 또는 회사 이름의 사용을 설명용으로만 사용되며 미국 정부에 의한 승인을 의미하지는 않습니다.

1. 글라스 울 준비 <ol><li> 필터의 각 일괄 처리 전에시키고 이후 10 %의 표백제 용액으로 작업 영역을 소독. </li><li> 장갑과 가운 입어. 최소한 20 분 동안 121 ° C와 15 PSI에 autoclaving하여 양동이를 소독. 멸균 양동이에 유리 양모를 놓습니다. </li><li> 역삼투 물로 글라스 울을 포화 및 15 분 동안 흠뻑 젖어 보자. </li><li> 양동이에서 역삼투 물을 배수. </li><li> 1 M HCL로 글라스 울을 포화 및 15 분 동안 흠뻑 젖어 보자. </li><li> 양동이에서 1 M HCL 드레인. </li><li> 역삼투 물로 글라스 울을 씻어. </li><li> 철저하게 섞는다. </li><li> 산도 용지를 사용하여 산도를 확인하고 중성 산도가 달성될 때까지 역삼투 물 린스 반복한다. </li><li> 헹굼 물을 붓고. </li><li> 1 M NaOH로 글라스 울을 포화 및 15 분 동안 흠뻑 젖어 보자. </li><li> 양동이에서 1 M NaOH 드레인. </li><li> R중성 산도가 달성되기 전까지 역삼투 린스 epeat. </li><li> 헹굼 물을 붓고. </li><li> 대신 양동이로, 글라스 울 세척기는 디자인 유리 피펫 세척기 (그림 1)과 비슷있는 건설 수 있습니다. </li><li> 멸균 인산과 완전히 유리 양모를 맡아 식염수 (PBS)는 산도를 6.8로 조정 버퍼. </li><li> 4에서 준비한 유리 즉시 양털 또는 상점을 사용 ° C. 그것은 최대 2 주 동안 사용자에게 위해 저장할 수 있습니다. 사용하기 전에, 그것이 시간이지나면서 상승하므로 산도 중립한지 확인하십시오. 산도가 중성이 아닌 경우, 멸균 인산과 다시 헹굼 (산도 6.8) 염분 버퍼. </li></ol> 2. 유리 양모 필터 조립 <ol><li> 남성 어댑터 나일론 피팅은 대문자로 엉망 수 있도록 PVC 모자와 탭 스레드로 16분의 11 인치 구멍을 드릴 다운합니다. 이 단계는 첫 번째 어셈블리를 위해 꼭 필요한 사항입니다. 이후 마개는 년간 사용하실 수 있습니다. 테플론을 적용나일론 피팅 스레드와 뚜껑의 나사에 테이프. </li><li> 글라스 울 작은 조각으로 PVC 파이프를 싸. 꽉를 싣고, 자동차 엔진 밸브와 같은 금속 플런저를 사용하십시오. 포장은 큰 힘을 필요로하지 않습니다. 꽉 충분한 글라스 울은 제자리에 유지되도록 싸서 채널을 형성하지 않습니다. 그러나 너무 단단히 물이 필터를 통해 흐를 수를 포장하지 않습니다. 적절하게 포장되면 분당 4-5리터의 유속은 필터가 40-60 PSI 사이의 압력으로 물을 도청에 부착되었을 때 달성해야합니다. 이 프로토콜에 명시된 PVC 파이프의 크기의 경우 약 85g은 씻어과 포장 글라스 울은 파이프마다 사용됩니다. 포장 재료의 중량은 파이프 질량 85 그램 한물간 글라스 울로 최고로드 균형과 팩을에 빈 PVC 파이프 증가 때까지. </li><li> 첨부 남성 어댑터 나일론 피팅과 PVC 마개로 폴리 프로필렌 메쉬를 삽입합니다. </li><li> PVC 파이프의 나사 부분에 테플론 테이프를 적용합니다. </li><li> PVC p로 PVC 모자에 나사IPE 및 레이블 하나의 필터 끝에 유입하고 다른 쪽 끝을 유출. 이것은 용출 단계 나중에 중요하다. 어떤 끝이 유입 상관 없어요 표시됩니다. </li><li> 멸균 인산의 60 ML은 카테터를 사용하여 필터로 (산도 = 6.8)을 식염수 버퍼 푸시는 주사기 줬지. 초과는 반대 끝이 나올 것이다. </li><li> 포장은 누수 방지하기 Parafilm과 밀접하게 끝납니다. 필터는 4 ° C.에 30 일 이내에 저장될 수 </li></ol> 3. 샘플링 <ol><li> 0.525 % NaClO (즉, 일반 가정용 표백제의 10 % 용액)에 30 분간 항목을 순환하거나 immersing하여 샘플링에 사용되는 튜브를 소독. 차아 염소 산염 용액을 배수하여이 작업을 따라 역삼투 물의 일리터에 2 %의 주식 무수 나트륨 thiosulfate 용액의 25 ML을 추가하여 만든 0.05 % 나트륨 thiosulfate로 무력화. </li><li> 7.5보다 큰 산도와 견본 물의 경우 HCL과 6.5 사이 7.0으로 산도를 조정합니다. HCL 농도 범위는 수 0.25에서한 M. 포 리터에 M 0.5 M HCL은 일반적으로 물이 주변 산도와 버퍼링 용량에 따라 두 800리터 샘플, 충분합니다. 연동 펌프 또는 Venturi을 (그림 2)를 사용하여 샘​​플링 기간 동안 HCL을 주사. 펌프 속도 또는 대상 산도를 달성하기위한 Venturi 개구부를 조정합니다. 필드 산도 측정기를 사용하여 샘​​플 라인 콘센트에서 산도를 측정합니다. </li><li> 글라스 울 필터 나막신 경우 높은 탁도와 물에서 prefilter을 사용합니다. (prefilter과 주택 사양 자료 목록을 온라인에 있습니다.) 병원균은 그것이 (아래) 유리 양모 필터와 함께 eluted해야 prefilter에 의해 갇힌 입자에 장착할 수 있기 때문에. </li><li> 둘 사이 4리터 / 분으로 유량을 조정합니다. 일반 샘플 볼륨 200 사이 1천5백리터 있습니다. </li><li> 최대 48 시간 동안 4 ° C에서 플라스틱 무균 봉투에 유리 양모 필터와 저장소를 분리합니다. </li></ol> 4. 용리 <ol><li> 접사 링에 유리 양모 필터샘플 유입 엔드 폴리 프로필렌 병으로 하향 지적과 함께하라. 샘플 흐름의 방향을 반대로 Elute. </li><li> 필터로 9.5의 산도와 0.05 M 글리신에 무균 3 % 쇠고기 추출물의 80 ML 밀어. </li><li> 15 분 정도 기다리십시오. </li><li> 필터로 9.5의 산도와 0.05 M 글리신에 무균 3 % 쇠고기 추출물의 또 다른 80 ML 밀어. </li><li> 거품이 필터 입구에서 나오는 공기까지 철저한 필터를 누르십시오. </li><li> 1 M HCL과 7.0 사이 7.5에 eluate의 산도를 조정합니다. </li><li> 매장 4에 eluate ° C에서 시간의 긴 기간 동안 최대 24 시간까지 또는 -20 ° C에서. </li><li> 하나를 사용하는 경우 prefilter을 Elute. 나사가 빠지다가 주택 카트리지의 상단과 멸균 장갑 낀 손으로 장소에 prefilter을 누른 상태 하우징 내부에 모두 잔류 물을 부어. </li><li> 주택 카트리지에서 prefilter를 제거하고 15 &quot;X 6&quot;지퍼 잠금 가방에 그것을 풀. </li><li> 0.05 M g의 무균 3 % 쇠고기 추출물 200 ML을 부어가방에 9.5의 산도와 지퍼 잠금 장치로 안전하게 도장 lycine. </li><li> 전체 표면 쇠고기 추출물과 접촉되도록 빌어 prefilter를 여러 번 반전. </li><li> 15 분 기다려 가끔씩 반전하고 빌어 prefilter을 마사지. </li><li> 지퍼 잠금 장치를 엽니다. prefilter 주변 그립 가방이 꽉 가능한 한 많은 쇠고기 추출물로 짜낸하고, 멸균 장갑 낀 손으로 prefilter을 당겨합니다. </li><li> 폴리 프로필렌 병에 쇠고기 추출물의 eluate 하거라. </li><li> 1M HCL과 7.0 사이 7.5에 eluate의 산도를 조정합니다. </li><li> 매장 4에 eluate ° C에서 시간의 긴 기간 동안 최대 24 시간까지 또는 -20 ° C에서. prefilter의 eluate는 별도로 병원균에 대한 분석이나 유리 양모 필터 eluate과 결합 할 수 있습니다. </li></ol><s팬 클래스 = &quot;pdflinebreak&quot;&gt; 5. 대표 결과 <table border="1"><tbody><tr><td> 병원체 </td><td> 물의 탁도 수준 (NTU) </td><td> 금액 시드 / L B, C, D </td><td> % 복구 ± 1 SD </td><td> 숫자 독립 평가판 </td></tr><tr><td> Enterovirus-Poliovirus 세이빈 III </td><td> 0.5 </td><td> 500 </td><td> 81% ± 11 </td><td> 7 </td></tr><tr><td> 힘들어바이러스 Poliovirus 세이빈 III </td><td> 0.5 </td><td> 5000 </td><td> 67% ± 12 </td><td> 8 </td></tr><tr><td> Enterovirus-Poliovirus 세이빈 III </td><td> 215 </td><td> 500 </td><td> 59% ± 32 </td><td> 7 </td></tr><tr><td> Enterovirus-Poliovirus 세이빈 III </td><td> 215 </td><td> 5000 </td><td> 38% ± 22 </td><td> 6 </td></tr><tr><td> Enterovirus-Poliovirus 세이빈 III </td><td> 447 </td><td> 500 </td><td> 56% ± 18 </td><td> 8 </td></tr><tr><td> Enterovirus-Poliovirus 세이빈 III </td><td> 447 </td><td> 5000 </td><td> 63% ± 37 </td><td> 8 </td></tr><tr><td> Cryptosporidium parvum </td><td> 0.5 </td><td> 5 </td><td> 38% ± 14 </td><td> 7 </td></tr><tr><td> Cryptosporidium parvum </td><td> 0.5 </td><td> 50 </td><td> 53% ± 19 </td><td> 8 </td></tr><tr><td> 울어ptosporidium parvum </td><td> 215 </td><td> 5 </td><td> 40% ± 16 </td><td> 7 </td></tr><tr><td> Cryptosporidium parvum </td><td> 215 </td><td> 50 </td><td> 30% ± 6 </td><td> 6 </td></tr><tr><td> Cryptosporidium parvum </td><td> 447 </td><td> 5 </td><td> 33% ± 13 </td><td> 8 </td></tr><tr><td> Cryptosporidium parvum </td><td> 447 </td><td> 50 </td><td> 28% ± 11 </td><td> 8 </td></tr><tr><td> 살모넬라 enterica </td><td> 0.5 </td><td> 5 </td><td> 29% ± 24 </td><td> 7 </td></tr><tr><td> 살모넬라 enterica </td><td> 0.5 </td><td> 500 </td><td> 56% ± 16 </td><td> 8 </td></tr><tr><td> 살모넬라 enterica </td><td> 215 </td><td> 5 </td><td> 32% ± 24 </td><td> 7 </td></tr><tr><td> 살모넬라 enterica </TD&gt; <td> 215 </td><td> 500 </td><td> 34% ± 11 </td><td> 6 </td></tr><tr><td> 살모넬라 enterica </td><td> 447 </td><td> 5 </td><td> 34% ± 18 </td><td> 8 </td></tr><tr><td> 살모넬라 enterica </td><td> 447 </td><td> 500 </td><td> 31% ± 24 </td><td> 8 </td></tr></tbody></table><ol class="lalpha"><li> Nephelometric 탁도 단위 </li><li> 게놈 사본 / L로 qPCR가 열거 Enteroviruses </li><li> C. parvum은 oocysts로 immunofluorescence가 열거 </li><li> S. enterica는 식민지 - 성형 단위로 문화가 열거 </li></ol> 다양한 수상 turbidities 및 병원균 밀도와 표 1. 유리 양모 농도. <table border="1"><tbody><tr><td> 병원체 </td><td> 물 샘플 위치 </td><td> 금액 시드 / L </td><td> % Reco<html> 대단히 </td></tr><tr><td> 조류 독감 H5N2 </td><td> Sundi 호수, 앵커리지 자치구 </td><td> 2500 </td><td> 42.9 % </td></tr><tr><td> 조류 독감 H5N2 </td><td> Minto 플래 츠, 페어 뱅크스 노스 스타 자치구 </td><td> 2500 </td><td> 36.7 % </td></tr><tr><td> 조류 독감 H5N2 </td><td> 운반 밸리, 앵커리지 자치구 </td><td> 2500 </td><td> 7.8 % </td></tr><tr><td> 조류 독감 H5N2 </td><td> 포터 마쉬, 앵커리지 자치구 </td><td> 2500 </td><td> 41.5 % </td></tr><tr><td> 조류 독감 H5N2 </td><td> 윌로우 호수, 유콘-Koyukuk의 자치구 </td><td> 2500 </td><td> 15.5 % </td></tr></tbody></table><ol class="lalpha"><li> 게놈 사본 / L로 qPCR로 측정 </li></ol> 조류 독감 바이러스의 표 2. 유리 양모 농도는 알래스카에서 다섯 사이트에서 물을 사용합니다. les/ftp_upload/3930/3930fig1.jpg &quot;고도 =&quot;그림 1 &quot;/&gt; 그림 1. 글라스 울 와셔의 다이어그램. 이것은 rinsing에서 시간을 절약하는 대신 양동이에 사용될 수있다. 개념은 유리 피펫 세척기와 비슷합니다. <img src="/files/ftp_upload/3930/3930fig2.jpg" alt="그림 2" /> 연동 펌프에 의한 산성 주입과 그림 2. 유리 울 여과. 펌프 튜브가 물이 수돗물과 글라스울 필터 사이의 샘플 라인에 산성을 소개하는 &quot;T&quot;커넥터가 있습니다. 샘플 물이 산도에게&gt; 7.5가있는 경우에만 산도 조정이 필요합니다. 유리 양모 필터는 탁도 수준과 병원균 밀도 (표 1) 다양한으로 물로부터 병원균을 집중에 효과적입니다. 이것을 테스트하려면 dechlorinated 수돗물 20리터은 탁도의 원하는 수준을 달성하기위한 건조 미사 롬 토양 (0, 1.27, 또는 2.75 G / L)과 혼합하고 다양한 밀도의 병원균으로 놓는되었다. 물글라스 울 필터 통과되었다 eluate에 집중 병원균은 열거하고,이 값은 퍼센트 복구 계산에서 분자였다되었다. , %의 복구 계산의 분모입니다 물,,에 놓는 병원균의 양을 먼저 다음 병원체를 열거하는 부정적인 eluate로 병원균을 퍼뜨리고에 의해 결정되었다. 부정적인 eluate는 필터를 통해 unseeded 20리터 샘플을 전달하고 eluting에 의해 준비되었다. 부정적인 eluate에 놓는 병원균을 Quantifying 것은 유리 양모 필터에 의해 만들어진 매트릭스의 차이에서 발생할 수있는 병원균 열거의 차이가 발생하지 않습니다. qPCR에 의한 병원균을 quantifying이 단계의 중요성은 Lambertini 외에 설명되어 있습니다. 3. 20리터 부정적인 컨트롤 샘플 퍼센트 복구 계산을 먹이고 수도 네이티브 병원균의 존재가 없었 결정하는 글라스 울 여과를 통해 집중되었다. 10 μm의 공칭 기공 크기 prefilter w탁도 수준이 ≥ 215 NTU 때 같이 사용됩니다. Poliovirus은 Lambertini 외에 설명되어. 3 차 농축 및 핵산 추출 절차 및 primers와 프로브를 사용하여 실시간 qPCR에 의해 계량되었다. Cryptosporidium parvum은 poliovirus를위한 보조 농도 절차에서 만든 최종 농축 시료 부피 (FCSV) 논리적으로 양이되었다 . Oocysts은 immunofluorescence (MeriFluor Cryptosporidium 및 Giardia 탐지 키트, 메리디안 생명 과학 주식 회사, 신시내티, OH)에 의한 시각 있었다. 살모넬라 enterica는 XLD 한천로 도금하여 FCSV 논리적으로 양이 (Remel, Lenexa, KS) 및 집계되었다 식민지 - 성형 - 단위. 유리 양모 필터는 조류 독감 바이러스 (표 2) 집중에 효과적입니다. 낮은 pathogenicity 조류 독감 바이러스 (H5N2)는 알래스카의 여러 위치에서 물에 놓는 및 abov을 설명한대로 %의 복구 계산전자. 차 집중과 핵산 절차 poliovirus 대한으로 수행되었다; 바이러스 Spackman 외에 설명된 primers와 프로브를 사용 qPCR하여 계량되었다 4.

<ol>
	<li>US Environmental Protection Agency. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+1623:+Cryptosporidium+and+Giardia+in+Water+by+Filtration/IMS/FA.">Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA.</a> EPA 815-R-05-002. , (2012).</li><li>Cashdollar, J. L., Dahling, D. R. <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+a+method+to+re-use+electropositive+cartridge+filters+for+concentrating+viruses+from+tap+and+river+water.">Evaluation of a method to re-use electropositive cartridge filters for concentrating viruses from tap and river water.</a> J. Virol. Methods. 132, 13-17 (2006).</li><li>Lambertini, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+of+enteroviruses,+adenoviruses,+and+noroviruses+from+drinking+water+by+use+of+glass+wool+filters.">Concentration of enteroviruses, adenoviruses, and noroviruses from drinking water by use of glass wool filters.</a> Appl. Environ. Microbiol. 74, 2990-2996 (2008).</li><li>Spackman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+real-time+reverse+transcription+PCR+assay+for+Type+A+influenza+virus+and+the+avian+H5+and+H7+hemagglutinin+subtypes.">Development of a real-time reverse transcription PCR assay for Type A influenza virus and the avian H5 and H7 hemagglutinin subtypes.</a> J. Clin. Microbiol. 40, 3256-3260 (2002).</li><li>Environment Agency. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimisation+of+a+new+method+for+detection+of+viruses+in+groundwater.">Optimisation of a new method for detection of viruses in groundwater.</a> Report No. NC/99/40. , (2000).</li><li>Vilagin&#233;s, P., Sarrette, B., Husson, G., Vilaginés, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glass+wool+for+virus+concentration+at+ambient+water+pH+level.">Glass wool for virus concentration at ambient water pH level.</a> Water Sci. Technol. 27, 299-306 (1993).</li><li>Vivier, J. C., Ehlers, M. M., Grabow, W. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+enteroviruses+in+treated+drinking+water.">Detection of enteroviruses in treated drinking water.</a> Water Res. 38, 2699-2705 (2004).</li><li>Powell, K. L., Sililo, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Enteric virus detection in groundwater using a glass wool trap. In: Groundwater: Past Achievements and Future Challenges. , 813-816 (2000).</li><li>Hunt, R. J., Borchardt, M. A., Richards, K. D., Spencer, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+sewer+source+contamination+of+drinking+water+wells+using+tracers+and+human+enteric+viruses.">Assessment of sewer source contamination of drinking water wells using tracers and human enteric viruses.</a> Environ. Sci. Technol. 44, 7956-7963 (2010).</li><li>van Heerden, J., Ehlers, M. M., Heim, A., Grabow, W. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence,+quantification+and+typing+of+adenoviruses+detected+in+river+and+treated+drinking+water+in+South+Africa.">Prevalence, quantification and typing of adenoviruses detected in river and treated drinking water in South Africa.</a> J. Appl. Microbiol. 99, 234-242 (2005).</li><li>Vilagin&#233;s, P. <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+glass+wool+method+for+poliovirus+recovery+from+drinking+water+and+sea+water.">Round robin investigation of glass wool method for poliovirus recovery from drinking water and sea water.</a> Water Sci. Technol. 35, 445-449 (1997).</li><li>Gantzer, C., Senouci, S., Maul, A., Levi, Y., Schwartzbrod, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enterovirus+genomes+in+wastewater:+concentration+on+glass+wool+and+glass+powder+and+detection+by+RT-PCR.">Enterovirus genomes in wastewater: concentration on glass wool and glass powder and detection by RT-PCR.</a> J. Virol. Methods. 65, 265-271 (1997).</li><li>Borchardt, M. A., Jokela, W. E., Spencer, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+losses+in+surface+water+runoff+from+dairy+manure+applied+to+corn+fields.">Pathogen losses in surface water runoff from dairy manure applied to corn fields.</a> , (2011).</li><li>Deboosere, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+concentration+method+for+the+detection+of+influenza+A+viruses+from+large+volumes+of+surface+water.">Development and validation of a concentration method for the detection of influenza A viruses from large volumes of surface water.</a> Appl. Environ. Microbiol. 77, 3802-3808 (2011).</li><li>Lambertini, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+contamination+from+operation+and+maintenance+practices+in+small+drinking+water+distribution+systems.">Virus contamination from operation and maintenance practices in small drinking water distribution systems.</a> J. Water Health. 9, 799-812 (2011).</li></ol>

관심 분야의 어떠한 충돌 선언 없습니다.

유리 양모 필터와 같은 완료 식수 7 지하수 8,9, 표면 물 10, 바다 물 11, 폐수 12 등 물의 다양한 소스에서 인간 장용 바이러스를 집중 여러 연구 팀으로 3,5,6에서 사용하고있다 농업 결선 13. 여기 필터는 또한 조류 인플루엔자 바이러스뿐 아니라 세균 및 병원체 protozoan 각각 살모넬라 enterica (serovar Typhimurium)와 Cryptosporidium parvum을 ...

<table border="1"><tbody><tr><td> 시약 또는 항목의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 염산 </td><td> <a href="http://www.fishersci.com" target="_blank">피셔 과학</a> </td><td> A144-500 </td></tr><tr><td height="24"> 수산화 나트륨 </td><td> <a href="http://www.fishersci.com" target="_blank">피셔 과학</a> </td><td> BP359-212 </td></tr><tr><td> 인산염은 염분 버퍼 염화나트륨 (소금) 인산 칼륨 - 이염 인산 칼륨 - 일염기의 </td><td> <a href="http://www.fishersci.com" target="_blank">피셔 과학</a> <a href="http://www.fishersci.com" target="_blank">피셔 과학</a> <a href="http://www.fishersci.com" target="_blank">피셔 과학</a> </td><td><html> BP358-212 BP363-500 BP362-500 </td></tr><tr><td> 나트륨 차아 염소 산염 즉, 가정용 표백제 </td><td> <a href="http://www.clorox.com" target="_blank">Clorox (주)</a> </td><td></td></tr><tr><td> 나트륨 thiosulfate, 무수 </td><td> <a href="http://www.fishersci.com" target="_blank">피셔 과학</a> </td><td> S 475-212 </td></tr><tr><td> 쇠고기 추출물, desiccated </td><td> <a href="http://www.bd.com" target="_blank">Becton, 디킨스와 회사</a> </td><td> 211,520 </td></tr><tr><td> 글리신 </td><td> <a href="http://www.fishersci.com" target="_blank">피셔 과학</a> </td><td> G46-500 </td></tr><tr><td> 기름을 바른 sodocalcic 글라스울 또는 R-11 unfaced 유리 섬유 절연 </td><td> <a href="http://www.isover.com" target="_blank">Isover</a> <html> 심판 = &quot;http://www.jm.com&quot;대상 = &quot;_blank&quot;&gt; 존스 맨빌 </td><td> Bourre 725 QN </td></tr><tr><td> 폴리 프로필렌 메쉬 </td><td> <a href="http://www.industrialnetting.com" target="_blank">그물 산업</a> </td><td> xN4510 </td></tr><tr><td> 2 &quot;X4&quot;SCH 80 PVC 스레드 파이프 니플 </td><td> <a href="http://www.grainger.com" target="_blank">Grainger</a> </td><td> 6MW35 </td></tr><tr><td> 2 &quot;SCH 40 PVC 캡 </td><td> <a href="http://www.grainger.com" target="_blank">Grainger</a> </td><td> 5WDW3 </td></tr><tr><td> 남성 어댑터 나일론 피팅 (1 / 2 &quot;1 개 / 2&quot;) </td><td> <a href="http://www.usplastic.com" target="_blank">미국 플라스틱 주식 회사</a> </td><td> 62,178 </td></tr><tr><td> eluate-1리터위한 샘플 병 </td><td> <a href="http://www.fishersci.com" target="_blank">피셔 과학</a> </td><td> 03-313 - 4 층 </td></tr><tr><td> 60 ML의 주사기 </td><td>&quot;http://www.fishersci.com&quot;대상 = &quot;_blank&quot;&gt; 피셔 과학 </td><td> NC9661991 </td></tr><tr><td> 산도 스트립 </td><td> <a href="http://www.whatman.com" target="_blank">워트 먼지</a> </td><td> 2614 991 </td></tr><tr><td> Prefilter, 폴리 프로필렌, 10 인치 카트리지, 10 μm의 </td><td> McMaster-카 </td><td> 4411K75 </td></tr><tr><td> Prefilter 주택 </td><td> 콜 - Parmer </td><td> S-29820-10 </td></tr></tbody></table>

glass wool filters for concentrating waterborne viruses and agricultural zoonotic pathogens

의심 오염된 물에 병원체 수준을 평가의 중요한 첫 번째 단계는 농도입니다. 농도 방법은 예를 들어, 샘플링 프로그램이 하나 이상의 병원체 그룹을 대상으로하는 경우 여러 가지 방법이 필요합니다 의미한다 Giardia와 Cryptosporidium 1에 대한 미국 환경 보호청 방법 1623 특정 병원체 그룹에 대한 구체적인 경향이 있습니다. 현재 방법의 또 다른 단점은 장비 예를 들어, 바이러스에게 2 집중에 대한 1MDS 카트리지 필터 VIRADEL 방법 복잡하고 비쌀 수있다. 이 문서에서는 waterborne 병원균을 집중을위한 글라스 울 필터를 구성하는 방법에 대해 설명합니다. 필터는 용출 후 원액은 문화적 또는 분자 방법에 의해 병원균 감지 및 열거 다음과 같은 원심 분리와 같은 두 번째 농도 단계, 의무가있다. 필터는 몇 가지 장점이 있습니다. 건설 간단하고 필터를 만들 수 있습니다특정 샘플링 요구 사항을 충족을위한 NY 크기. 필터 부분은 가능한 심하게 프로젝트 예산에 영향을주지 않고 샘플의 다수를 수집하고, 저렴합니다. 대형 샘플 볼륨 (100s 1,000의 패)은 샘플 탁도에서 막힘의 비율에 따라 집중하실 수 있습니다. 필터는 매우 휴대용이며, 펌프 및 유량계 등 최소한의 장비로, 그들은 완성된 식수, 표면 물, 지하수 및 농업 결선을 샘플링을 위해 현장에서 구현할 수 있습니다. 마지막으로, 글라스 울 여과은 하나의 방법이 필요하므로 병원체 종류의 다양한 집중을위한 효과적입니다. 여기 waterborne 인간 enterovirus, S almonella enterica, Cryptosporidium parvum 및 조류 독감 바이러스를 자세하게 필터 효과에 대한 리포트입니다.

Watch this Scientific Journal Video about Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens at JoVE.com

Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens

유리 양모 필터는 전세계 연구 그룹의 숫자 waterborne 바이러스를 집중하는 데 사용되었습니다. 여기 글라스울 필터를 구축하기위한 간단한 접근 방식을 보여 주며 필터도 waterborne 바이러스성, 세균성 및 protozoan 병원균을 집중 효과입니다 보여줍니다.

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 ...

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JoVE Journal

Immunology and Infection

17.9K 조회수.  United States Geological Survey. 유리 양모 필터는 전세계 연구 그룹의 숫자 waterborne 바이러스를 집중하는 데 사용되었습니다. 여기 글라스울 필터를 구축하기위한 간단한 접근 방식을 보여 주며 필터도 waterborne 바이러스성, 세균성 및 protozoan 병원균을 집중 효과입니다 보여줍니다.

비디오: Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1. A tick-borne murine model 2 has been developed to study Lyme disease in the laboratory. While na&iacute;ve ticks can be infected with B. burgdorferi by feeding them on infected mice, the molting process takes several weeks to months to complete. Therefore, development of more rapid and efficient tick infection techniques, such as a microinjection-based procedure, is an important tool for the study of Lyme disease 3,4. The procedure requires only hours to generate infected ticks and allows control over the delivery of equal quantities of spirochetes in a cohort of ticks. This is particularly important as the generation of B. burgdorferi infected ticks by the natural feeding process using mice fails to ensure 100% infection rate and potentially results in variation of pathogen burden amongst fed ticks. Furthermore, microinjection can be used to infect ticks with B. burgdorferi isolates in cases where an attenuated strain is unable to establish infection in mice and thus can not be naturally acquired by ticks 5. This technique can also be used to deliver a variety of other biological materials into ticks, for example, specific antibodies or double stranded RNA 6. In this article, we will demonstrate the microinjection of nymphal ticks with in vitro-grown B. burgdorferi. We will also describe a method for localization of Lyme disease pathogens in the tick gut using confocal immunofluorescence microscopy.

Lyme disease research studies often require generation of ticks infected with the pathogen Borrelia burgdorferi, a process that typically takes several weeks. Here we demonstrate a microinjection-based tick infection procedure that can be accomplished within hours. We also demonstrate an immunofluorescence method for in situ localization of B. burgdorferi within ticks.

틱 굿 내에서 빠른 전송과 라임 질병 병원균의 현지화을위한 방법

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1.  ...

연구

면역학 및 감염병학

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

카테고리의 감지 세균성 병원균에 대한 &#39;발광 생물&#39;리포터 파지

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Avian Influenza Surveillance with FTA  Cards: Field Methods, Biosafety, and Transportation Issues Solved

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral subtypes2. Human pandemics of flu originally stem from AIVs3. Many fatal human cases during the H5N1 outbreaks in recent years were reported. Lately, a new AIV related strain swept through the human population, causing the 'swine flu epidemic'4. Although human trading and transportation activity seems to be responsible for the spread of highly pathogenic strains5, dispersal can also partly be attributed to wild birds6, 7. However, the actual reservoir of all AIV strains is wild birds.
In reaction to this and in face of severe commercial losses in the poultry industry, large surveillance programs have been implemented globally to collect information on the ecology of AIVs, and to install early warning systems to detect certain highly pathogenic strains8-12. Traditional virological methods require viruses to be intact and cultivated before analysis. This necessitates strict cold chains with deep freezers and heavy biosafety procedures to be in place during transport. Long-term surveillance is therefore usually restricted to a few field stations close to well equipped laboratories. Remote areas cannot be sampled unless logistically cumbersome procedures are implemented. These problems have been recognised13, 14 and the use of alternative storage and transport strategies investigated (alcohols or guanidine)15-17. Recently, Kraus et al.18 introduced a method to collect, store and transport AIV samples, based on a special filter paper. FTA cards19 preserve RNA on a dry storage basis20 and render pathogens inactive upon contact21. This study showed that FTA cards can be used to detect AIV RNA in reverse-transcription PCR and that the resulting cDNA could be sequenced and virus genes and determined.
In the study of Kraus et al.18 a laboratory isolate of AIV was used, and samples were handled individually. In the extension presented here, faecal samples from wild birds from the duck trap at the Ottenby Bird Observatory (SE Sweden) were tested directly to illustrate the usefulness of the methods under field conditions. Catching of ducks and sample collection by cloacal swabs is demonstrated. The current protocol includes up-scaling of the work flow from single tube handling to a 96-well design. Although less sensitive than the traditional methods, the method of FTA cards provides an excellent supplement to large surveillance schemes. It allows collection and analysis of samples from anywhere in the world, without the need to maintaining a cool chain or safety regulations with respect to shipping of hazardous reagents, such as alcohol or guanidine.

A method to preserve, detect and sequence RNA from Avian Influenza Viruses was validated and extended using natural faecal samples from birds. This technique removes the necessity of maintaining a cool chain and handling of infectious viruses and can be applied in a 96-well high-throughput setup.

조류 FTA 카드 인플루엔자 감시 : 필드 방법, Biosafety, 그리고 교통 문제 해결

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral ...

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

베로 세포 배양 분석하여 필드 수집 모기에서 감염성 바이러스의 검출

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ixodes Scapularis 진드기의 타액, 침샘 및 Hemolymph 컬렉션

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

양극화 인간기도 액체 덮인 문화를 구축 호흡기 병원균에 대한 숙주 세포 응답을 공부하는 상피 Calu-3 전지<em&gt; 체외에서</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA 바이러스의 넓은 스펙트럼 화학 억제를위한 높은 처리량 검사

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

키메라 마우스 모델에서 재조합 인플루엔자 백신의 비강 관리는 점막 면역을 연구하기 위해

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described. Second, a method is provided for the adsorption of compounds containing long-chain hydrocarbons to convert non-fusogenic glass into a fusogenic substrate. Third, this protocol describes fabrication of surface micropatterns that promote a high degree of spatiotemporal control over MGC formation. Finally, fabricating glass bottom dishes is described. Examples of use of this in vitro cell system as a model to study macrophage fusion and MGC formation are shown.

This protocol describes the fabrication of optical-quality glass surfaces adsorbed with compounds containing long-chain hydrocarbons that can be used to monitor macrophage fusion of living specimens and enables super-resolution microscopy of fixed specimens.

대 식 세포 융합 연구 품질 광학 유리 표면 조작

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging ...

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.

Here, we present an immunohistochemistry test protocol for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues.

포르말린 고정 조직에서 리사바이러스 항원 검출을 위한 면역조직화학 시험

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. ...