我们感谢 Susanne. 拉斯穆森和 Solvej Kolbjørn 詹森为他们的技术援助和处理临床样品。该项目是 EbolaMoDRAD 财团的一部分, 该集团已从创新医学倡议2根据赠款协议 N°115843的联合承诺获得资金。这项联合承诺得到欧洲联盟2020地平线研究和创新计划和 EFPIA 的支持。

资本区域生物医学研究伦理学委员会已给予知情同意, 此处描述的所有方法均获豁免, 不受道德委员会制度的审查, 这是根据丹麦关于化验发展项目的法律。
1. 用于病毒灭活和快速 NA 提取的采血真空管的研制
注意: 此协议使用的缓冲区包含胍硫氰酸盐 (GITC) 和非离子表面活性剂, 这是刺激物。采取适当的实验室安全措施, 使用流动罩, 并在处理时戴上手套。避免皮肤和眼睛接触。如果缓冲区溢出, 污染的表面绝不能直接与胺或次氯酸钠 ("漂白剂" 中的活性成分) 进行消毒, 因为这种混合物可能导致有毒氰化物的形成。首先, 用吸收性组织擦拭溢出的缓冲器。接下来, 用70% 乙醇和水清洗表面, 最后使用胺或次氯酸钠。
<ol><li>要准备采血真空管, 将1.6 毫升的特定商业裂解缓冲液注入4毫升 EDTA 真空管, 用25克 x 1 针和3毫升注射器刺穿管盖。 注: 请勿拆卸真空管盖。必须保持真空。</li>
	<li>将含有缓冲液的真空管贮存在室温下, 直至使用。 注: 该管在制备后至少1年稳定。</li>
</ol>2. 制备 NA 萃取缓冲器
<ol><li>要准备用于 NA 提取的缓冲器, 请在机架中放置两个1.8 毫升、两个4.5 毫升和一个3.6 毫升管。</li>
	<li>添加, 由吹打, 960 µL 磁性玻璃微粒 (MGPs) 到一个干净的1.8 毫升管和标签它与 MGPs. 并用重悬赛车暂停完全之前吹打它。 注: MGPs 往往迅速收集在底部的管。</li>
	<li>添加, 由吹打, 4 毫升的洗涤缓冲器 I 到一个干净的4.5 毫升管和标签它为 WB-1。 注意: 洗涤缓冲器我含有胍氯, 这是刺激性的。采取适当的实验室安全措施, 使用流动罩, 并在处理时戴上手套。避免皮肤和眼睛接触。</li>
	<li>添加, 由吹打, 1.5 毫升洗涤缓冲 II 到一个干净的3.6 毫升管和标签它为 WB-2。</li>
	<li>添加, 由吹打, 3 毫升洗涤缓冲 III 到一个干净的4.5 毫升管和标签它为 WB-3。</li>
	<li>添加, 由吹打, 100 µL 洗脱缓冲器到一个干净的1.8 毫升管和标签为 EB。</li>
	<li>将 aliquoted 缓冲区存储在室温下, 直到使用。 注: 该管在制备后至少1月内稳定。</li>
</ol>3. 病毒感染体征和症状患者的血液收集
注意: 在收集病人的全血时, 采取适当的实验室安全措施。戴上手套和眼镜。如果病人是孤立的, 请遵循生物安全4级程序。
<ol><li>要收集病人的静脉全血, 使用蝶针与小孔延长管和血液收集真空管含有裂解/捆绑缓冲。把病人的手臂放在向下的位置, 把收集管放在比蝶针低的位置。将蝶形针插入病人的静脉内, 并将采血真空管连接至小孔延伸。 注: 这将防止回流。</li>
	<li>血液收集后, 通过翻转管 5-10 倍, 混合的管的内容。 注: 由于血液收集管中含有1.6 毫升溶解/结合缓冲液的残余真空, 采集的样品体积将自动为1.6 毫升。</li>
	<li>用70% 乙醇消毒管外。</li>
	<li>室温下将试管孵化20分钟。 注: 该协议可以暂停在这里, 完整的采血管可以储存在-20 °c, 5 °c, 25 °c 或37°c 至少1月7。</li>
	<li>继续直接到简化 NA 提取方法。</li>
</ol>4. 全血的简化 NA 提取
<ol><li>用手 5-10x 翻转真空管, 将钠从裂解/捆绑缓冲液中纯化出来, 并将该管的内容混合在一起。</li>
	<li>小心拆卸管盖, 并排出盖子。</li>
	<li>将准备好的整除 MGPs (1 毫升) 直接倒入采血管中。</li>
	<li>在含有该样本的管上放置一个未使用的采血管的新盖子。</li>
	<li>把手指放在盖子上, 以确保管子紧紧地闭合, 并用手翻转采血管 5-10 倍来混合管的内容。</li>
	<li>把管子放在磁性支架上, 把手指放在盖子上, 以确保管子紧闭。</li>
	<li>用手轻轻翻转磁性持有者几次, 以确保所有的 MGPs 都收集在管的一侧与磁铁。 注: 磁性支架可以用细长磁铁和橡皮筋代替。</li>
	<li>去掉管子的盖子, 用一次性吸管或简单地将内容倒入50毫升的收集管中, 丢弃管子的内容。 注: 用盖子关闭管, 避免50毫升收集管中的气溶胶。</li>
	<li>将准备好的整除 WB-1 (4 毫升) 直接倒入采血管中。</li>
	<li>把盖子放在管子上, 把手指放在盖子上, 以确保管子紧闭。</li>
	<li>把管子从磁支架上取下, 用手指把盖子牢牢地拧紧。 注: 如果使用磁铁和橡皮筋, 只需从管中取下橡皮带和磁铁即可。</li>
	<li>并用重悬 MGPs 通过手翻转采血管 5-10 倍。</li>
	<li>重复步骤 4.6-4.8。</li>
	<li>将准备好的整除 WB-2 (1.5 毫升) 直接倒入采血管中。</li>
	<li>把盖子放在管子上, 把管子从磁支架上取下。 注: 如果使用磁铁和橡皮筋, 只需从管中取下橡皮带和磁铁即可。</li>
	<li>把一根手指放在盖子上, 以确保管子紧闭。</li>
	<li>并用重悬 MGPs 通过手动翻转血液收集管几秒钟。</li>
	<li>重复步骤 4.6-4.8。</li>
	<li>将准备好的整除 WB-3 (3 毫升) 直接倒入采血管中。</li>
	<li>把盖子放在管子上, 把管子从磁支架上取下。 注: 如果使用磁铁和橡皮筋, 只需从管中取下橡皮带和磁铁即可。</li>
	<li>把一根手指放在盖子上, 以确保管子紧闭。</li>
	<li>并用重悬 MGPs 通过手翻转采血管 5-10 倍。</li>
	<li>重复步骤 4.6-4.8。</li>
	<li>将 EB (100 µL) 的制备整除直接倒入采血管中。</li>
	<li>把盖子放在管子上, 把管子从磁支架上取下。 注: 如果使用磁铁和橡皮筋, 只需从管中取下橡皮带和磁铁即可。</li>
	<li>并用重悬用手指敲击采血管 5-10 次, 在 EB 中 MGPs。 注意: 该协议可以在这里暂停, 管可以存储在-20 摄氏度。</li>
	<li>将悬浮 MGPs 的一滴 (5-8 µL) 转移到下游 NA 放大反应混合使用1.5 毫升一次性吸管 (任何下游诊断 NA 放大试验, 如灯/rt 灯或 qPCR/rt-qPCR 化验可以使用)。 注: NAs 将坚持 MGPs, 所以一定要使用 MGPs 在下游 NA 放大反应。使用前混合赛车悬浮液。混合后, MGPs 将收集在管的底部。</li>
</ol>

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作者没有什么可透露的。

在本报告中, 我们描述了一种安全、快速、简单的人工床边 NA 提取方法, 用于在裂解/捆绑缓冲-灭活全血中对病毒进行下游分子检测。所述的 NA 提取方法是直接对含有裂解/捆绑缓冲 (材料表) 的真空采血管收集的全血标本进行的.7。此特定缓冲区钝化 EBOV7 , 是该方法中的关键组件。血液收集后, 我们建议孵化管至少20分钟7 , 以确保完...

在病毒暴发的情况下, 当病人被限制在医院隔离病房或需要快速诊断时, 安全、简单、准确的点对诊的分子诊断是必要的病人护理和风险遏制。最近两次病毒暴发, 在西非的埃博拉病毒 (EBOV) 和南美的 Zika 病毒 (2015), 增加了对改进的护理点分子诊断测试的兴趣, 如反向转录回路介导的等温放大 (RT 灯)1,2和 recombinase 聚合酶放大 (RPA)3,4。RT 灯和 RPA 都是快速、敏感和特定的分子测试, 可以在简化的样品制备中进行。对 Zika 病毒, 将 RT 灯与侧流法 (LFA) 相结合, 在30分钟内检测非纯化全血标本中的 Zika 病毒;然而, 对于被归类为风险组4病原体并具有高度传染性的 EBOV, 需要在生物安全等级 4 (BSL-4) 条件下处理和灭活, 然后才能进行任何安全诊断程序。
EBOV 的简化失活方法, 例如在2、3、4、5的样品中加入裂解缓冲剂, 在暴发期间使用;然而, 这些方法需要在 BSL-4 条件下处理与实验室设备, 如 BSL-3 生物安全柜, 离心机, 暖气块, 吸管, 至少。这种设备通常不存在于隔离病房或外地医院。为了克服这一挑战, 已尝试在手提箱3中进行诊断, 并开发了一些便携设备和机器 (例如, 一种用于核酸 (NA) 提取的便携设备)6。然而, 在使用这些设备之前, EBOV 阳性样品仍需进行灭活。
我们以前报告了一个快速的床边病毒灭活方法为 EBOV7, 痘苗病毒和牛痘病毒8通过增加一个商业裂解/捆绑缓冲到普通的真空采血管, 允许直接血液从患者转移到灭活缓冲器7。这种直接和直接的钝化在一个封闭的系统, 消除了处理样品使用任何严格的遏制, 如 BSL-4 条件7, 并在正常 BSL-2 条件下处理样品的需要。这种灭活方法与几种 NA 萃取系统兼容, 如机器人和手净化套件7;然而, 这些方法需要实验室设备, 如机器人, 离心机, 和电力, 这并不总是存在于现场设置或在医院隔离病房。
在本报告中, 我们描述了一种安全、快速和简化的人工 NA 提取方法, 用于随后在裂解/捆绑缓冲-灭活全血中的病毒分子检测。NA 萃取方法不需要任何设备, 除了磁铁/磁性持有人。NA 萃取不需要离心机、加热块或电。因此, 这种方法不依赖于实验室设施, 而且可以很容易地用于任何地方 (如在野战医院、医院隔离病房或低资源环境下)。na 萃取方法快速简便, 可直接用于任何下游 NA 试验, 如 qPCR、rt qPCR、灯或 rt 灯。当该 NA 提取方法与灯和便携式电池驱动的等温仪相结合时, 可以在40分钟内采集到血液中的床边诊断。

<table><tbody><tr><td>Vacutainer K2 EDTA tubes (4 mL)</td><td>Becton Dickinson</td><td>368861</td><td></td></tr><tr><td>Plus Blood Collection</td><td>Becton Dickinson</td><td>362725</td><td></td></tr><tr><td>23G x 1 needle</td><td>Becton Dickinson</td><td>300800</td><td></td></tr><tr><td>LUER LOK syringe (3 mL)</td><td>Becton Dickinson</td><td>309658</td><td></td></tr><tr><td>Butterfly needle with small-bore extension tubing</td><td>J&amp;oslash;rgen Kruuse A/S</td><td>121714</td><td></td></tr><tr><td>1% Virkon</td><td>Nomeco A/S</td><td>849265</td><td></td></tr><tr><td>MagNA Pure LC DNA Isolation Kit I - Lysis/Binding Buffer - Refill</td><td>Roche Diagnostics A/S</td><td>03246752001</td><td></td></tr><tr><td>MagNA Pure LC Total Nucleic Acid Isolation Kit</td><td>Roche Diagnostics A/S</td><td>3038505001</td><td></td></tr><tr><td>Nunc&amp;trade; Biobanking and Cell Culture Cryogenic Tubes (1,8 mL)</td><td>Thermo Fisher Scientific</td><td>375418</td><td></td></tr><tr><td>Nunc&amp;trade; Biobanking and Cell Culture Cryogenic Tubes (3,6 mL)</td><td>Thermo Fisher Scientific</td><td>379189</td><td></td></tr><tr><td>Nunc&amp;trade; Biobanking and Cell Culture Cryogenic Tubes (4,5 mL)</td><td>Thermo Fisher Scientific</td><td>379146</td><td></td></tr><tr><td>DynaMag&amp;trade;-5 Magnet</td><td>Thermo Fisher Scientific</td><td>12303D</td><td></td></tr><tr><td>Transfer pipette (3.5 mL)</td><td>Sarstedt</td><td>86.1171.010</td><td></td></tr><tr><td>Thermo Scientific&amp;trade; Samco&amp;trade; Fine Tip Transfer Pipettes (1.5 mL)</td><td>Thermo Fisher Scientific</td><td>231</td><td></td></tr><tr><td>Thermo Scientific&amp;trade; Nunc&amp;trade; 50 mL Conical Sterile Polypropylene Centrifuge Tubes</td><td>Thermo Fisher Scientific</td><td>339652</td><td></td></tr></tbody></table>

rapid, safe, and simple manual bedside nucleic acid extraction for the detection of virus in whole blood samples

对感染的快速诊断对于暴发管理、风险控制和病人护理至关重要。我们以前已经显示了一种方法, 快速床边的埃博拉病毒在血液取样的安全核酸 (NA) 测试通过添加一个商业裂解/捆绑缓冲直接进入真空采血管。利用这种床边灭活方法, 我们开发了一种安全、快速、简便的床边 NA 提取方法, 用于随后在裂解/捆绑缓冲-灭活全血中检测病毒。钠萃取直接在采血管中进行, 无需设备或电。
将血液收集到溶解/结合缓冲液中后, 通过手工翻转管将其内容混合, 并在室温下孵化出20分钟的混合物。将磁性玻璃微粒 (MGPs) 添加到管中, 通过手工翻转收集管将其内容混合。MGPs, 然后收集在血液收集管的一侧使用磁性持有人或磁铁和橡皮筋。MGPs 清洗三次, 在将洗脱缓冲器直接添加到采集管后, NAs 就可以进行 NA 测试, 如 qPCR 或等温环放大 (灯管), 而不需要从反应中去除 MGPs。NA 提取方法不依赖任何实验室设施, 可以很容易地在任何地方使用 (例如, 在外地医院和医院隔离病房)。当该 NA 提取方法与灯和便携式仪器相结合时, 可在采血40分钟内获得诊断。

本文提出的协议简单有效, 可广泛应用于用裂解/结合缓冲剂灭活的传染性全血标本进行的任何分子检测。血液失活和 NA 提取工作流程如图 1所示, 包括采血真空管的制备7 (图 1A), 采血7 (图 1B), NA 提取 (图 1C - 1H) 和下游 NA 分析 (图 ...

Watch this Scientific Journal Video about Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples at JoVE.com

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

在这里, 我们提出了一个快速病毒核酸提取从病毒灭活全血的协议。萃取是直接在采血管, 不需要设备或电力。该方法不依赖于实验室设施, 可以在任何地方使用 (例如在野战医院)。

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

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

rapid-safe-simple-manual-bedside-nucleic-acid-extraction-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

7.3K 次观看.  Statens Serum Institut. 在这里, 我们提出了一个快速病毒核酸提取从病毒灭活全血的协议。萃取是直接在采血管, 不需要设备或电力。该方法不依赖于实验室设施, 可以在任何地方使用 (例如在野战医院)。

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

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

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

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。第三部分。病毒检测用RT-qPCR的

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

科研

环境科学

Preparation of Viral DNA from Nucleocapsids

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

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

从核衣壳的病毒DNA的制备

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

免疫与感染

High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively).

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. Consequently, the sample preparation format is compatible with most liquid handling instruments, and can be used for many medium to high-throughput clinical applications and sample types.

高通量，自动化的DNA和RNA的提取从临床样品使用TruTip技术常见的液体处理机器人

TruTip is a simple nucleic acid extraction  technology whereby a porous, monolithic binding matrix is inserted into a  pipette tip. The geometry of the ...

生物工程

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

纳米测序全血 mRNA 测序

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

遗传学

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

用阴离子交换树脂检测 Bioaerosols 病毒

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

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

病毒 DNA 的纯化及相关病毒和细胞蛋白的鉴定

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from clinical and biological sources. The method is unbiased and universal; it uses random primers for cDNA synthesis and requires no prior knowledge of the viral sequence content. Before library construction, selective RNase H-based digestion is used to deplete unwanted RNA &#8212; including poly(rA) carrier and ribosomal RNA &#8212; from the viral RNA sample. Selective depletion improves both the data quality and the number of unique reads in viral RNA sequencing libraries. Moreover, a transposase-based &#39;tagmentation&#39; step is used in the protocol as it reduces overall library construction time. The protocol has enabled rapid deep sequencing of over 600 Lassa and Ebola virus samples-including collections from both blood and tissue isolates-and is broadly applicable to other microbial genomics studies.

This protocol describes a rapid and broadly applicable method for unbiased RNA-sequencing of viral samples from human clinical isolates.

临床样品RNA病毒的无偏深度测序

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from ...

医学

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

粗病毒提取中 DNA 的组合分析及对感染叶 RNA 的分析发现新的病毒基因组

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent.
At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes.
The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of use.

病毒测试,水样中检测病毒的一种使用点方法

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤:一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...