이 작품은 국립 의학 연구위원회, 싱가포르에 의해 재정 지원되었다.

1. 뎅그열 바이러스의 알렉사 플루어 라벨링 <ol><li> 라벨 반응하기 전에, 자당 쿠션과 뎅그열 바이러스를 정화하고 같은 프로토콜에 표시된 필요한 시약과 장비를 준비합니다. </li><li> 라벨 및 필터 주사기 0.2μm 필터를 소독 바로 앞에 신선한 0.2M 나트륨 중탄산염 버퍼, pH를 8.5 (라벨 버퍼) 및 히드록 실 아민 1.5M 버퍼, 산도 8.3을 (시약을 중지) 준비합니다. </li><li> 2ml 튜브에 버퍼를 라벨링의 1ml에 뎅그열 바이러스를 정화 약 3x10 8 플라크 형성 단위 (pfu)를 희석. 이것은 바이러스의 일괄 라벨에 대한 비례까지 확장할 수 있습니다. </li><li> Reconstitute 동결 건조된 알렉사 플루어 594 (AF594) 전에 라벨 반응 즉시 버퍼를 라벨에 1mM로 succinimidyl 에스테르. 알렉사 플루어 염료 시리즈에서 다른 fluorochromes 하나의 필요에 따라 사용할 수 있습니다. 이 단계 이후 조명에 노출을 최소화합니다. </li><li> 피펫 팁로 부드럽게 교반하면서 희석 바이러스에 1mM AF594 염료의 100μl를 추가합니다. </li><li> 어둠 속에서 1 시간을 위해 실내 온도에서 상표 반응 믹스를 품어. 부드러운 뒤집어 모든 15mins로 섞는다. </li><li> 테이블탑 원심 분리기에서 튜브 잠시 돌려 및 피펫 팁로 부드럽게 교반하면서 반응 혼합물을 중지 시약의 100μl를 추가합니다. </li><li> 어둠 속에서 추가 시간 동안 실온에서 품어. 부드러운 뒤집어 모든 15mins로 섞는다. </li></ol> 2. 퓨리화잉 알렉사 플루어는 뎅그열 바이러스를 분류 <ol><li> 그 동안에, 선택의 버퍼와 정화 열 평형. 본 실험에서는 PD - 10 컬럼 HNE 버퍼의 25ml (5mM Hepes, 150mM NaCl, 0.1mM EDTA (에틸렌 다이아 민 테트라 초산)), 사용하기 전에 산도 7.4과 equilibrated 있습니다. </li><li> 칼럼의 맨 위에 표시된 바이러스를 적용하고 표시된 바이러스가 매트릭스를 입력 한때를 통해 흐름을 모으기 시작합니다. 모든 레이블이 붙은 바이러스가 매트릭스를 입력한 후에는 HNE 버퍼로 열을 채웁니다. 흐름을 통해 첫 2.5ml를 버리고 표시된 바이러스 분율의 다음 2ml를 수집합니다. </li><li> -80에서 AF594 표시 뎅그열 바이러스를 정화 나누어지는하고 저장 ° C, 거리 광원에서. </li></ol><ol><li> 한 나누어지는를 해동 및 레이블 바이러스의 배치를 사용하기 전에 상패 분석​​에 의해 표시된 바이러스의 titer를 결정한다. </li><li> 감염 전에 잘 하루의 하단에 coverslip과 4 잘 판에서 M - 199 성장 매체 재배 종자 베로 세포의 5x10 4 당 음,. </li><li> 37 우물에 문화의 표면에 뜨는를 제거하고 10 분 대한 100μl 볼륨 (필요에 따라 M - 199 유지 보수 매체 희석)에 표시 바이러스 1 감염의 다중성으로 세포를 감염 ° C. </li><li> inoculums를 제거하고 1xPBS에 두 coverslips 씻으십시오. </li><li> 30 분 3 %의 paraformalydehyde에있는 세포를 수정. </li><li> 1xPBS에 coverslips 3 번 씻으십시오. </li><li> 30 분 대한 1xPBS에서 0.1 %의 사포닌 및 5 % BSA가 포함된 permeabilization 솔루션 세포 Permeabilize. </li><li> 로 세포를 품어 undiluted 원심 분리 - 명확히, 습기 챔버에서 1 시간에 대한 단클론 항체 3H5의 하이 브리 도마 문화 뜨는 빛으로부터 보호. </li><li> (1xPBS는 1mM의 염화칼슘, 1mM 마그네슘 염화물 및 0.1 %의 사포닌을 포함) 씻어 버퍼에 coverslips 3 번 씻으십시오. </li><li> AF488 안티 - 마우스 IgG 항체와 세포를 품어, 1:100은 빛으로부터 보호 습한 실내에 45mins를위한, permeabilization 솔루션 희석. </li><li> 세척 버퍼에 coverslips 3 번 씻고 탈이온수에 한 번 씻어. </li><li> DAB 8μl Mowiol 4-88 포함된 2.5 %의 Dabco로 초과 물을 배수하고 유리 슬라이드에 마운트 종이 타월에 대한 coverslip의 가장자리. </li><li> 자이스 혈구 공촛점 현미경을 사용하여 볼 전에 4 하룻밤 설정 장착 솔루션 ° C를 허용합니다. 순차 인수는 concomitantly 감지기 사이의 시간과 스위칭에 흥미로운 한 형광단을 수행하여야한다. </li><li> 이미지는 다음 라벨의 정도를 추정하기 위해 자이스 혈구 LSM 선 (禅) 소프트웨어를 사용하여 표시된 바이러스와 E 단백질 항체 얼룩의 공동 지방화에 대해 분석하고 있습니다. </li></ol> 3. 대표 결과 AF594 염색으로 표시 뎅그열 바이러스의 수율의 예제는 그림 2에 표시됩니다. 일반적으로 초기 titer 10 배 드롭 미만은 성공적인 라벨링 다음과 같은 관찰해야합니다. 그러나, 모든 버퍼가 성공하고 그들이 수성 솔루션 8 nonreactive 무료 지방산으로 hydrolyze로 알렉사 플루어 succinimidyl의 에스테르가 reconstitution 즉시 사용해야 라벨에 대한 신선한 준비하는 것을인지해야합니다. 다음, 라벨이 바이러스는 실험에 사용하기 전에 충분한 형광 검사되어야한다. 간단한 immunofluorescence 분석은 베로 세포에서 수행되었으며 라벨의 정도는 안티 - E 단백질 항체 얼룩으로 표시된 바이러스의 공동 지방화의 추정 수 있습니다. 끊다앨 세포 검사되었으며 전형적인 공촛점 이미지가 그림 3에 표시됩니다. virions 약 65-80%이 염료와 함께 레이블 것을 제안, 0.65에서 0.8에 이르기까지 계수를 중복 보여주 LSM 선종 소프트웨어를 사용하여 이미지의 공동 지방화 분석. <img src="/files/ftp_upload/3168/3168fig1.jpg" alt="그림 1" /> 그림 1.Overall 체계는 뎅그열 바이러스 절차의 알렉사 플루어 염료 라벨을 묘사한. 첫째, 해당 버퍼 및 정화 뎅그열 바이러스가 준비하고 있습니다. 알렉사 플루어 염료는 재구성하고 버퍼를 라벨에 희석 뎅그열 바이러스에 추가됩니다. 반응은 다음 중지 시약의 추가 1 시간 후에 중지됩니다. 이후, 레이블 바이러스 무료 염료를 제거하는 크기 제외 칼럼을 통해 정화됩니다. 마지막으로, 표시된 바이러스는 플라크의 분석에 의해 다시 titrated와 형광을위한 테스트입니다. <img src="/files/ftp_upload/3168/3168fig2.jpg" alt="그림 2" /> 그림 2. 로 AF594 분류 전후 상패 분석 결정 가능한 virions (pfu / ML)의 번호를 의미합니다. AF594 표시 뎅그열 바이러스의 나누어지는는 해동 후 다시 titrated 상패 분석에 의해 그것은 일반적으로 이하의 10 배 드롭로부터 표시합니다 시작 titer. 오류 막대는 중복의 표준 편차를 나타냅니다. <img src="/files/ftp_upload/3168/3168fig3.jpg" alt="그림 3" /> 그림 3. 이전 일 coverslips에 성장 베로 세포에서 뎅그열 바이러스 E 단백질과 AF594 레이블의 공동 지방화. 전지는 37 10 분 1 뫄에서 AF594 표시된 뎅그열 감염된 ° C. 세포는 그 후에 고정 및 안티 - E 항체로 분류하고, E 단백질의 colocalization (녹색) 및 AF594 라벨 (적색)을 검사했다. 형광 신호는 자이스 혈구 LSM 710 공촛점 현미경을 사용하여 63X 배율에 따라 시각되었습니다. 스케일 바 10μm이다. 삽입된 페이지의 그림과 같이 노란색은 colocalization의 영역을 나타냅니다.

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AF594 염료가이 보고서에 사용했지만, 알렉사 플루어 succinimidyl의 에스테르 시리즈 fluorophores 다양한 유사한 상표 화학 사용할 수 있습니다. 이 영상 이외 라벨 응용 프로그램을 확장할 수 있습니다. 유동세포계측법는 흥분과 외과 기계가 감지할 수 fluorophores에 대한 상표의 정도를 계산하는 공촛점 현미경의 대안으로 사용할 수 있습니다. 알렉사 플루어의 염료는 무료 아미노 ?...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> 나트륨 중탄산염 </td><td> 시그마 - 알드리치 </td><td> S6297 </td><td></td></tr><tr><td> 히드록 실 아민 </td><td> 시그마 - 알드리치 </td><td> 159417 </td><td></td></tr><tr><td> 수산화 나트륨 </td><td> 머크 </td><td> 106498 </td><td></td></tr><tr><td> AF594 succinimidyl 에스테르 </td><td> 분자 프로브, Invitrogen </td><td> A20004 </td><td></td></tr><tr><td> PD - 10 컬럼 </td><td> GE 헬스케어 </td><td> 17-0851-01 </td><td></td></tr><tr><td> Hepes </td><td> 시그마 - 알드리치 </td><td> H6147 </td><td></td></tr><tr><td> NaCl </td><td> 시그마 - 알드리치 </td><td> S3014 </td><td></td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) </td><td> 시그마 - 알드리치 </td><td> E9884 </td><td></td></tr><tr><td> M - 199 </td><td> Invitrogen </td><td> 11,150 </td><td></td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30070.03 </td><td></td></tr><tr><td> 4 - 잘 플레이트 </td><td> Nunc </td><td> 176740 </td><td></td></tr><tr><td> Coverslips </td><td> Einst </td><td> 0,111,520 </td><td></td></tr><tr><td> 현미경 슬라이드 </td><td> 브랜드 세일 </td><td> 7105 </td><td></td></tr><tr><td> 3H5 하이 브리 도마 </td><td> ATCC </td><td> HB46 </td><td></td></tr><tr><td> 10X PBS </td><td> 첫번째 자료 </td><td> BUF - 2040 - 10X1L </td><td></td></tr><tr><td> 사포닌 </td><td> 시그마 - 알드리치 </td><td> S4521 </td><td></td></tr><tr><td> BSA </td><td> 시그마 - 알드리치 </td><td> A7906 </td><td></td></tr><tr><td> 마그네슘 염화물 </td><td> 시그마 - 알드리치 </td><td> M2670 </td><td></td></tr><tr><td> 염화칼슘 </td><td> 시그마 - 알드리치 </td><td> C3306 </td><td></td></tr><tr><td> Paraformaldehye </td><td> 시그마 - 알드리치 </td><td> 15,812-7 </td><td></td></tr><tr><td> Mowiol 4-88 </td><td> Calbiochem </td><td> 475904 </td><td></td></tr><tr><td> Dabco </td><td> 시그마 - 알드리치 </td><td> D27802 </td><td></td></tr><tr><td> 테이블탑 원심 분리기 </td><td> Eppendorf </td><td> 5424 </td><td></td></tr><tr><td> 공촛점 현미경 </td><td> 자이스 혈구 </td><td> LSM 710 </td><td></td></tr></tbody></table> M - 199 성장 매체를 준비하려면, 나트륨 pyruvate와 M - 199, 살균 필터의 500ml에 중요하지 않은 아미노산 5ml의 5ml, FBS의 50ml를 추가합니다. M - 199 유지 보수 매체를 준비하려면, FBS의 15ml 나트륨 pyruvate와 M - 199, 살균 필터의 500ml에 중요하지 않은 아미노산 5ml의 5ml를 추가합니다.

visualizing dengue virus through alexa fluor labeling

바이러스와 세포 사이의 상호 작용의 초기 이벤트는 감염의 결과에 지대한 영향을 가질 수 있습니다. 이 상호 작용에 영향을 미치는 요인을 결정하는 것은 질병 pathogenesis 개선 이해로 이어질 때문에 예방이나 치료 설계에 영향을 미칠 수 있습니다. 따라서,이 상호 작용을 조사하는 방법의 개발이 유용할 것입니다. fluorophores 개발 1-3 및 이미징 기술 4 최근 발전은 뎅그열 pathogenesis에 대한 현재의 지식을 개선하기 때문에 매년 발생하는 뎅그열 감염의 수백만을 줄이기 위해 방법을 포장에 이용하실 수 있습니다. 싸여 뎅그열 바이러스는 단일 가닥 RNA 긍정적인 게놈 5가 들어있는 nucleocapsid 껍질을 보호 90 봉투 당단백질 (E) dimers 구성된 외부 비계를 타고있다. 바이러스 표면에 동일한 단백질 subunits 따라서 아민 반응 염료로 분류하고 immunofluorescent 현미경을 통해 시각 수 있습니다. 여기, 우리는 알렉사 플루어 succinimidyl 에스테르 염색가 라벨 후 높은 가능한 바이러스를 굴복 나트륨 중탄산염 버퍼에 직접 용해와 뎅그열 바이러스의 라벨링의 간단한 방법을 제시한다. 라이브 바이러스 및 단백질 라벨은 보통 디메틸 sulfoxide의 색소의 reconstitution을 필요로 기존 제조 업체의 프로토콜의 라벨에 대한 표준화된 절차가 없습니다. 디메틸 sulfoxide의 존재, 심지어 분 수량에 바이러스의 생산 감염을 차단하고 또한 세포 세포 독성 6 유발 수 있습니다. 이 프로토콜의 디메틸 sulfoxide의 사용의 배제 따라서이 가능성을 감소. 알렉사 플루어의 염료가 우수한 photostability을 가지고 있으며 세포 이해와 바이러스의 endosomal 교통 연구에 이상적, 낮은 산도에 민감한 등 플루오레신와 rhodamine이 같은 일반적인 염료보다됩니다. 알렉사 플루어 염료의 활용은 바이러스 특정 항체 및 호스트 세포 7의 putative 수용체에 의해 표시 뎅그열 바이러스의 인식에 영향을주지 않았다. 이 방법은 virological 공부에 유용한 응용 프로그램을 수 있습니다.

Watch this Scientific Journal Video about Visualizing Dengue Virus through Alexa Fluor Labeling at JoVE.com

Visualizing Dengue Virus through Alexa Fluor Labeling

형광단 개발 및 이미징 기술, 바이러스가 바이러스와 세포 사이의 초기 상호 작용을 시각화하기 위해 고안되었다 뎅그열의 알렉사 플루어 라벨링의 간단한 방법에있는 발전을 활용.

알렉사 플루어 라벨링을 통해 뎅그열 바이러스를 떠올리

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

visualizing-dengue-virus-through-alexa-fluor-labeling

Research

JoVE Journal

Immunology and Infection

13.7K 조회수.  DSO National Laboratories. 형광단 개발 및 이미징 기술, 바이러스가 바이러스와 세포 사이의 초기 상호 작용을 시각화하기 위해 고안되었다 뎅그열의 알렉사 플루어 라벨링의 간단한 방법에있는 발전을 활용.

비디오: Visualizing Dengue Virus through Alexa Fluor Labeling

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

호중구의 분리 및 분석 치료제에 림프종 세포 감도에서 자신의 역할을 확인하는 방법

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

연구

면역학 및 감염병학

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

유전자 또는 약물 치료에 사용하기 위해 HIV-1 생산을 표적 RNA를의 효능 및 독성 평가

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

새로운 세계 Zika 바이러스 전염성 클론으로부터 재조합 바이러스의 구조 및 특성 규명

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

Confocal 현미경 검사 법을 사용 하 여 대 식 세포 세포 외 트랩을 시각화

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

간단한 Cytometry 결정 체 외에서 항 체 의존의 향상 뎅기열 바이러스 Zika 바이러스 미리 혈 청을 사용 하 여 분석 결과 기반으로

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

전체 혈액 샘플에서 바이러스의 검출, 안전, 신속 하 고 간단한 수동 침대 핵 산 추출

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.

실시간 정량 중 합 효소 연쇄 반응에 의해 감염 된 세포의 문화 상쾌한에 뎅 그 열 바이러스 RNA를 측정

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

코딩 및 비 코딩 RNA 클래스의 식별 표시 돼지에서 전 혈

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is characterized by lethal complications due to plasma leakage, ascites, pleural effusion, respiratory distress, severe bleeding, and organ impairment. A few cases of DENV infection present neurological manifestations; however, studies have not explored DENV-induced neuropathogenesis further. In this study, we present a protocol to use an immunocompetent outbred ICR (Institute of Cancer Research) mouse for investigating the induction of central nervous system (CNS) infection with DENV, followed by the progression of acute viral encephalitis-like disease.

Here, we present a protocol for creating an immunocompetent ICR (Institute of Cancer Research) murine model of central nervous system infection to display the development of neuropathy. Monitoring acute viral encephalitic disorders by identical disease scores could be performed for showing dengue virus-induced neuropathy in vivo.

뎅기열 바이러스 유발 급성 바이러스 뇌염 같은 질병의 뮤린 모델

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

광견병 백신 효능을 평가하기 위한 시험관 내 ELISA 테스트

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Methods Article

알렉사 플루어 라벨링을 통해 뎅그열 바이러스를 떠올리