우리는 원고의 중요한 읽기에 대한 박사 개밥 바라기 철 마리 라모스 감사드립니다. 이 작품은 연구 자원에 대한 국립 센터 (P20 RR0115635-6, C06 RR17417-01 및 G20RR024001)과 국립 신경 질환 연구소와 스트로크 (2R01 NS052609)에 의해 지원되었다.

1. 장비 준비 <ol><li> Misonix 3000이나 37 일정 온도를 유지하기 위해 열 전자 Neslab EX-7 물 목욕 (이케아, NH)에 연결 Misonix 4000 sonicator (Farmingdale, NY) ° C.를 사용하여 열 과학 (Waltham, MA)에서 얻은 돔 캡 200 μl 얇은 벽 PCR 관 스트립의 샘플을 Sonicate. </li><li> 새로운 sonicator는 지속적인 작동 9 &#39;브레이크에서 &quot;기간이 필요합니다. 두 달간 브레이크의 기간은 10로 설정 진폭 수준 40 초 sonication 버스트 10 분 부화 사이클 구성되어 있습니다. 하나는 티타늄 microplate 뿔 (그림 1, 패널 B)의 표면에 걸쳐 침식을 준수해야합니다. 순환 물은 티타늄 microplate 뿔의 침식으로 인해 흰 유탁을 찾습니다. 매일이 물을 교체합니다. </li><li> PRP SC의 증폭은 microplate의 뿔에 걸쳐 다양합니다. 가장 PRP SC 증폭 효율에서 구할 수 있습니다microplate 뿔 (그림 1, 패널 C 참조)의 중심에서 5cm의 반경입니다. </li><li> PMCA 중 하나 라운드는 144 사이클을 구성되어 있습니다. 각주기는 5 초 sonication과 10 분 부화로 구성됩니다. 이 약 PMCA의 원형 당 24 시간에 수반합니다. </li><li> sonicator의 제어판에 표시 sonication의 전원 출력은, sonicator의 랙과 물 목욕에서 순환 물 온도에 존재하는 PCR 튜브의 수에 따라 다릅니다. 물이 37 ° C에서 equilibrated 있는지 확인하고, sonicator의 튜브 랙 (그림 1, 패널 C)의 약 30 % 이상을 입력하지 않습니다. microplate의 뿔을 통해 PCR 튜브를 분산. PCR 관 스트립 사이의 빈 공간의 적어도 하나의 행을 가지고 (그림 1, 패널 C) 스트립 당 서로 연결 더 이상 사 이상 튜브를 할 수 있습니다. </li><li> sonication의 힘은 성공적으로 prion 변형 증폭을위한 중요합니다. sonicating 동안 전원 출력을 가지고 진폭을 조정155 ~ 170w 사이에 이르기까지. 저희 연구실에서 전력 수준은 각각 여섯 Misonix 3000 및 Misonix 4000 7로 설정되어 있습니다. </li><li> 하드 물 얼룩을 축적 방지하기 위해 물 욕조에 증류수를 사용합니다. 이 물 주간를 교체합니다. 부화와 sonication 사이클 동안, 물 응축의 microplate 통 뚜껑 안쪽에 발생합니다. 그림 1, 패널 A. 같이 결로를 방지하려면 sonicator의 인클로저 상자의 상단에 작은 수족관 열 패드를 부착 </li></ol> 2. 샘플 준비 <ol><li> 동물을 포함한 모든 절차가 크 레이튼 대학 기관 동물 케어의 승인을위원회를 사용하여 실험 동물의 관리 및 사용에 대한 가이드를 준수했다. 조직의 수집하기 전에 동물이 anesthetized 및 transcardially 얼음처럼 차가운 인산염을 사용하여 perfused해야합니다은 뇌에서 피를 제거하고의 억제를 방지하기 위해 산도 7.4에서 5 MM EDTA (에틸렌 다이아 민 테트라 초산)과 생리 식염수를 버퍼혈액에 7 PMCA 반응. 무균 기술을 사용하여 빠른 속도로 동물 조직을 수집하고 각 prion 변형을위한 전용 분해 도구를 제공합니다. 절개 후, 1.5 ML의 microcentrifuge 관에 조직을 배치, 플래시 드라이 아이스에 고정하고, -80에서 보관 ° C를 균질화까지. </li><li> PMCA 기판 (PRP C) 솔루션을 준비하려면 Tenbroeck 조직 분쇄기를 사용하여 얼음 차가운 변환 버퍼와 10 %의 중량 / 부피 (w / V) 솔루션 (3 부 참조)에 감염되지 않은 햄스터 뇌를 (UN BH) 균질화. homogenizing 있지만, 솔루션의 거품 과도한 방지하기 위해 그라인더 내부에 공기를 얻을하지 마십시오. 30 초와 나누어지는 1.5 ML의 microcentrifuge 튜브의 표면에 뜨는 500 XG에 centrifuging하여 솔루션을 명확히하십시오. -80에서 저장 ° C 더 사용까지. </li><li> PMCA 씨 (PRP SC) 솔루션을 준비하려면, 얼음 차가운 wate하는 10 % w / V 솔루션으로 전염성 물질 (뇌, 비장, 림프절, 다른 조직이나 오염 된 토양)을 균질화0.6 ML의 microcentrifuge 튜브의 연구와 나누어지는 솔루션입니다. -80에서 저장 ° C 더 사용까지. </li></ol> 3. 변환 버퍼를 준비 <ol><li> 50 ML의 용적 플라스크 (결과는 트라이 튼 X-100의 1 % 최종 농도)을 (; Steinheim, 독일 시그마 - 알드리치 GmbH의)에서 트리톤 X-100의 0.5093 g 무게. </li><li> 하나의 완전한 단백 분해 효소 억제제 태블릿 (, 만하임, 독일 로슈 진단 GmbH가)를 추가합니다. </li><li> EDTA (에틸렌 다이아 민 테트라 초산) 0.0931 g (이 EDTA (에틸렌 다이아 민 테트라 초산) 5 MM 최종 농도가 높아집니다) (, Phillipsburg, NJ Mallinckrodt 베이커 주식회사)를 추가합니다. </li><li> 1N NaOH를 7.4로 산도를 조정합니다. </li><li> Dulbecco의 인산과 50 ML에 볼륨을 조정합니다 (, Manassas, VA Mediatech 주식회사) (DPBS) 식염 버퍼링. </li><li> 진공 여과 장치와 0.45 μm의 니트로 셀룰로스 막 통해 솔루션을 필터링 할 수 있습니다. 변환 버퍼는 더 이상보다 달 동안 4 ° C에 저장할 수 있습니다. </li></ol> 4. PMCA를 사용 Prion의 변종의 증폭 <ol><li>PCR 튜브에 1시 20분의 비율 (PMCA 씨와 PMCA 기판의 95 μl의 예 혼합 5 μl)에 PMCA 기판에 PMCA의 씨앗을 희석. -80에서 15 μl 나누어지는을 제거하고 저장 ° unsonicated 제어를위한 C,이 unsonicated 제어는 서양 얼룩 (WB) 분석에 의해 다음 proteinase K (PK) 소화를 통해 PRP SC 증폭을 수량화하는 데 사용됩니다. 추가 unsonicated 제어는 PMCA 반응의 기간 동안 37 ° C에서 두 번째 15 μl 나누어지는을 배치에 의해 생성 될 수 있습니다. 세 반복의 최소 통계 목적을 위해 필요합니다. </li><li> 제목 그림 1에 설명 된대로 PMCA 중 하나 라운드에 샘플의 나머지 (70 μl), 패널 A. 흔들어서는 튜브 PCR 튜브의 돔 뚜껑에 물 응축을 방지하기 위해 매 5 시간입니다. </li><li> 샘플의 튜브는 아래 돌다가 부드럽게 homog 보장 할 절차 후 열기 전에 (솔루션 돔 모자에 가서하지 않는 것이 조심하는) vortexed해야eneity하고 잠재적 인 오염을 피할 수 있습니다. </li><li> 짧은 잠복기의 변종 (예 : HY TME, HaCWD, 또는 263K)의 시리얼 PMCA (sPMCA)의 경우, 신선한 감염되지 않은 95 μl로 라운드에서 sonicated 샘플의 5 μl를 전송하여 1시 20분의 비율에 sonicated 자료를 희석 뇌 homogenate (그림 2, 패널 B). </li><li> 긴 잠복기의 변종 (예 : DY TME, 139H, 22CH, 22AH, 또는 ME7H)의 sPMCA를 들어, 신선한 감염되지 않은 50 μl로 라운드에서 sonicated 샘플의 50 μl를 전송하여 1시 1분의 비율에 sonicated 자료를 희석 뇌 homogenate (그림 2, 패널 B). </li><li> 이전에 희석 sonicated 혼합물 (단계 4.4 또는 4.5)에서 두 15 μl aliquots를 제거하고 -80에서 둘 중 하나를 저장하는 37 번 ° C와 다른 ° C (PMCA 2 회전 unsonicated 제어 솔루션). PMCA 두번째로 생물 될 수 있습니다. </li><li> 이 절차는 무한정 반복 할 수 있습니다. 저희 연구실에서는, 우리는변경되지 않은 나머지 변형 특성 15 PMCA 라운드까지 수행했습니다. </li><li> PK는 샘플을 소화. 전형적인 WB의 경우, 5 샘플의 PK 80 μg / ML 5 μl (이 솔루션은 40 μg / ML의 최종 PK 농도를해야합니다)의 μl 한 시간 동안 37 ° C에서 배양을 섞는다. 겔로드 버퍼의 10 μl를 추가합니다. 10 분에이 솔루션을 끓일. 솔루션은 냉각과 젤에 10 μl를로드 보자. </li><li> 증폭의 성공을 계산하는 것은 하나에 비교하여 증폭 PRP SC의 양을 추정하기 위해 PK-소화 샘플에 WB 분석을 수행 unsonicated 컨트롤 20,21 그림 2, 패널 C 또는 알려진 금액으로는 재조합 PK-소화되지 않은 PRP. </li></ol> 5. 교차 오염을 방지 중요한 단계는 교차 오염 및 단백 분해 효소 방지 제품의 드 노보 형성으로 인해 잘못된 반응의 발생을 최소화하기 위해 이동해야합니다. </P&gt; <ol><li> 각 prion 변형에 대한 샘플 수집을 위해 분해 도구 전용 세트를 (즉, 집게, 핀셋, 가위)를 사용하십시오. </li><li> PMCA 기판으로 사용할 감염되지 않은 머리를 homogenizing하기 전에, 1 %의 표백제와 피펫을 닦으십시오. 10 분에 1 %의 표백제와 sonicator의 PCR 튜브 랙, 조직 homogenizers, 그리고 PCR 튜브 스토리지 랙을 만끽하고 증류수로 철저히 씻어. </li><li> 신선한 Versi - 드라이 랩 뱅이 새로운 샘플 균질 할 수있는 모든 시간과 작업 공간을 커버, 새로운 PMCA 실험이 준비되어야하는 때 또는 sPMCA 라운드 사이에 aliquoting. </li><li> 새로운 장갑에게 새로운 샘플 (sPMCA 라운드 사이의 aliquots를 전송할 특히) 처리 할 때마다 사용합니다. </li><li> 각 PMCA주기에 짧은 sonication의 폭발을 사용합니다. 10 분 부화와 함께 5 초에서 sonication의 사이클이 드 노보 PRP SC 1,20,21,23,24를 생성하지 않고 prions를 증폭하기에 충분한 것입니다. 다른 연구소가 게재 한 바와 같이, 증가sonication 시간은, sonication의 진폭을 증가하고, PMCA 사이클의 수를 확장하는 데 노보 PRP SC 형성 2에 대한 가능성을 증가시킵니다. </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+sources+of+scrapie+prions.">Environmental sources of scrapie prions.</a> J. Virol. 84, 11560-11562 (2010).</li><li>Nichols, T. A., Pulford, B., Wyckoff, A. C., Meyerett, C., Michel, B., Gertig, K., Hoover, E. A., Jewell, J. E., Telling, G. C., Zabel, M. D. <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+protease-resistant+cervid+prion+protein+in+water+from+a+CWD-endemic+area.">Detection of protease-resistant cervid prion protein in water from a CWD-endemic area.</a> Prion. 3, 171-183 (2009).</li><li>Prusiner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+proteinaceous+infectious+particles+cause+scrapie.">Novel proteinaceous infectious particles cause scrapie.</a> Science. 216, 136-144 (1982).</li><li>Saborio, G. P., Permanne, B., Soto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+detection+of+pathological+prion+protein+by+cyclic+amplification+of+protein+misfolding.">Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding.</a> Nature. 411, 810-813 (2001).</li><li>Saunders, S. E., Bartz, J. C., Vercauteren, K. C., Bartelt-Hunt, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzymatic+treatment+of+soil-bound+prions+effectively+inhibits+replication.">An enzymatic treatment of soil-bound prions effectively inhibits replication.</a> Appl. Environ. Microbiol. 77, 4313-4317 (2011).</li><li>Saunders, S. E., Shikiya, R. A., Langenfeld, K., Bartelt-Hunt, S. L., Bartz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+efficiency+of+soil-bound+prions+varies+with+soil+type.">Replication efficiency of soil-bound prions varies with soil type.</a> Journal of virology. , (2011).</li><li>Schutt, C. R., Bartz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+interference+with+multiple+prion+isolates.">Prion interference with multiple prion isolates.</a> Prion. 2, 61-63 (2008).</li><li>Shikiya, R. A., Ayers, J. I., Schutt, C. R., Kincaid, A. E., Bartz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-infecting+prion+strains+compete+for+a+limiting+cellular+resource.">Co-infecting prion strains compete for a limiting cellular resource.</a> Journal of. 84, 5706-5714 (2010).</li><li>Shikiya, R. A., Bartz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+generation+of+high+titer+prions.">In vitro generation of high titer prions.</a> Journal of virology. , (2011).</li><li>Weber, P., Giese, A., Piening, N., Mitteregger, G., Thomzig, A., Beekes, M., Kretzschmar, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+genuine+prion+infectivity+by+serial+PMCA.">Generation of genuine prion infectivity by serial PMCA.</a> Veterinary microbiology. 123, 346-357 (2007).</li></ol>

관심 없음 충돌이 선언 없습니다.

증폭기 전염성 prion 단백질의 과제는 생체 실험의 긴 잠복 기간 및 비용입니다. PMCA 기술은 전염성 prion 요원을 증폭 할 수있는 비용 효과적인 수단입니다. 여러 실험실 정확하게 체외 7, 9, 12, 14, 19,24에 prion의 긴장을 증폭 할 수 PMCA의 능력을 확인하고 있습니다. Prion 질환은 종 사이에 전염 될 수 있습니다. Bessen 및 습지 효과적으로 두 개의 햄스터 ?...

<table border="1"><tbody><tr><td> 시약 / 장비 </td><td> 제조업 자 </td><td> 고양이. 수 </td></tr><tr><td> Misonix 3000 </td><td> Misonix </td><td> S-3000 </td></tr><tr><td> Misonix 4000 </td><td> Misonix </td><td> S-4000 </td></tr><tr><td> Tenbroeck의 조직 그라인더 </td><td> Kontes </td><td> 885000-0007 </td></tr><tr><td> Neslab EX-7 물 목욕 </td><td> 열 전자 </td><td> Neslab EX-7 </td></tr><tr><td> 0.2 ML PCR 튜브 스트립 </td><td> 열 과학 </td><td> AB-0451 </td></tr><tr><td> 트리톤 X-100 </td><td> 시그마 알드리치 </td><td> T9284-100ML </td></tr><tr><td> 전체 프로테아제 억제제 </td><td> 로슈 </td><td> 11 697 498 001 </td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) </td><td> JT 베이커 </td><td> 4040-00 </td></tr><tr><td> DPBS </td><td> Mallinckrodt 베이커 Mediatech </td><td> 21-031 CV </td></tr><tr><td> Versi - 드라이 랩 Soakers </td><td> 피셔 과학 </td><td> 14 206 28 </td></tr><tr><td> Repti 칼로리 히터 </td><td> 동물원 메드 랩 주식회사 </td><td> RH-4 </td></tr></tbody></table>

protein misfolding cyclic amplification of prions

Prions 동물과 인간 9,18에 필연적으로 치명적인 전도 할 수있는 해면상 뇌병증 (TSE)을 일으킬 전염성 대리인입니다. prion 단백질은 두 개의 isoforms, 비 전염성 호스트 인코딩 단백질 (PRP C) 및 전염성 단백질 (PRP SC), PRP C 8 비정상적으로 - 접힌 이소 형이 있습니다. prion 에이전트 작업의 과제 중 하나는 이전의 호스트 접종 13 다음 임상 증상의 발전에 긴 잠복기입니다. 이 전통적으로 길고 비싼 동물 bioassay 연구를 위임. 또한, PRP SC의 생화학 및 biophysical 속성이 제대로 자신의 특이한 형태와 집계 상태로 인해 특징을 가지고 있습니다. PRP SC는 체외 14 PRP SC에 PRP C의 씨앗 변환 할 수 있습니다. PMCA은 adva 소요의 체외 기술이다PRP C와 PRP SC의 씨앗 19 분 금액을 초과하는 금액을 포함하는 시스템에서, 가속 속도로, PRP SC 많은 양의 생산 sonication과 부화주기를 사용하여이 능력을 ntage. 이 기술은 효과적으로 prion 변형 간섭을 에뮬레이션하기 위해 PRP C에서 PRP SC 전환의 종과 변형 특성을 요점을 되풀이하고, 감염된 조직, 체액, 환경 샘플 6,7,16에서 PRP SC의 매우 낮은 수준을 증폭하기 위해 증명되었습니다 23. , 오염을 최소화 일관된 결과를 생성하고, 그 결과를 정량화에 대한 권장 사항을 포함 본 논문은 내용 PMCA 프로토콜을. 우리는 또한 전염성 prion의 변종, prion 변형 간섭 및 환경에서 prions의 감지의 생성 및 특성 등 여러 PMCA 응용 프로그램을 논의합니다.

단백질 misfolding의 순환 증폭 (PMCA)는 체외 7 PRP SC, 12, 14, 19, 24을 증폭하는 데 사용됩니다. 성공적으로 PRP SC 증폭은 그림 3과 같이 PK 방지 prion 단백질 (햄스터 파생 prion의 변종이 19 ~ 30 kDa를 마이그레이션)의 서양 blots에 밴드 강도의 증가에 의해 표시됩니다.의 밴드 강도의 증가는 이후 PMCA는 PK 방지 PRP SC 소재의 증폭을 나타냅니다. ?...

Watch this Scientific Journal Video about Protein Misfolding Cyclic Amplification of Prions at JoVE.com

Protein Misfolding Cyclic Amplification of Prions

단백질 misfolding의 순환 증폭 (PMCA)는 prion 변환과 변형 및 종 장벽의 연구에서 시험 관내 분석이다. 또한 prion 검출 분석으로 사용할 수 있습니다.

Prions의 단백질 Misfolding의 순환 증폭

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

protein-misfolding-cyclic-amplification-of-prions

Research

JoVE Journal

Immunology and Infection

19.4K 조회수.  University of Nebraska at Lincoln. 단백질 misfolding의 순환 증폭 (PMCA)는 prion 변환과 변형 및 종 장벽의 연구에서 시험 관내 분석이다. 또한 prion 검출 분석으로 사용할 수 있습니다.

비디오: Protein Misfolding Cyclic Amplification of Prions

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

조류 종의 소화 기관을 통과 한 후 감염성 프리온을 확인하기위한 절차

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

연구

면역학 및 감염병학

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

다중 약물 내성을 보여<em&gt; 결핵균</em&gt; 증폭 마이크로 어레이

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

의 종 특이 검출을위한 루프 - 매개 등온 증폭 (LAMP) 분석 실험<em&gt; 에이 메리아</em&gt; 닭을 감염이

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

의 탐지를위한 동결 건조 루프 - 매개 등온 증폭 시약의 개발<em&gt; Coxiella burnetii의</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

실시간 떨게 유발 CWD Prions 지저분한 자료의 검색을 위한 변환 분석 결과

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

해 다중 단백질 신호 복합물 분자 보완성 친 화력 정화 (BiCAP)에 의해

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

자동화 된 행동 분석의 큰 C. 선 충 인구 넓은 시야의 사용 하 여 추적 플랫폼

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

생성, 증폭 및 재조합 호흡 Syncytial 바이러스의 적정

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the aquaculture industry that has resulted in the mass morbidity and mortality of tilapia in many parts of the world. An effective, rapid, and accurate diagnostic assay for TiLV infection is therefore necessary to detect the initial infection and to prevent the spread of the disease in aquaculture farming. In this study, a highly sensitive and practical reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is presented to detect tilapia lake virus in fish tissue. A comparison of the RT-qPCR and RT-LAMP assays of infected samples revealed positive results in 63 (100%) and 51 (80.95%) samples, respectively. Moreover, an analysis of uninfected samples showed that all 63 uninfected tissues yielded negative results for both the RT-qPCR and RT-LAMP assays. The cross-reactivity with five pathogens in tilapia was evaluated using RT-LAMP, and all the tests showed negative results. Both the liver and mucus samples obtained from infected fish showed comparable results using the RT-LAMP method, suggesting that mucus can be used in RT-LAMP as a nonlethal assay to avoid killing fish. In conclusion, the results demonstrated that the presented RT-LAMP assay provides an effective method for TiLV detection in tilapia tissue within 1 h. The method is therefore recommended as a screening tool on farms for the rapid diagnosis of TiLV.

Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus

We present an RT-LAMP assay for the detection of TiLV in tilapia fish using simple instruments over a relatively short period of time compared to conventional RT-PCR techniques. This protocol may help control the epidemic spread of TiLVD, especially in developing countries.

틸라피아 호수 바이러스의 특이적이고 신속한 검출을 위한 역전사 루프 매개 등온 증폭(RT-LAMP) 분석

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the ...

Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical regulators of necroptosis, an inflammatory form of cell death with important antiviral functions. Autophosphorylation of RIPK3 induces phosphorylation and activation of the pore-forming executioner protein of necroptosis MLKL. Trafficking and oligomerization of phosphorylated MLKL at the cell membrane results in cell lysis, characteristic of necroptotic cell death. The nucleic acid sensor ZBP1 is activated by binding to left-handed Z-form double-stranded RNA (Z-RNA) after infection with RNA and DNA viruses. ZBP1 activation restricts virus infection by inducing regulated cell death, including necroptosis, of infected host cells. Immunofluorescence microscopy permits the visualization of different signaling steps downstream of ZBP1-mediated necroptosis on a per-cell basis. However, the sensitivity of standard fluorescence microscopy, using current commercially available phospho-specific antibodies against human RIPK3 and MLKL, precludes reproducible imaging of these markers. Here, we describe an optimized staining procedure for serine (S) phosphorylated RIPK3 (S227) and MLKL (S358) in human HT-29 cells infected with herpes simplex virus 1 (HSV-1). The inclusion of a tyramide signal amplification (TSA) step in the immunofluorescent staining protocol allows the specific detection of S227 phosphorylated RIPK3. Moreover, TSA greatly increases the sensitivity of the detection of S358 phosphorylated MLKL. Together, this method enables the visualization of these two critical signaling events during the induction of ZBP1-induced necroptosis.

Tyramide signal amplification during immunofluorescent staining enables the sensitive detection of phosphorylated RIPK3 and MLKL during ZBP1-induced necroptosis after HSV-1 infection.