作者欣赏，莉萨彭慕兰格雷格史密斯，马特 - 莱​​曼，Marlies埃尔德里奇，Halina Staniszewska Goraczniak和Enquist实验室成员在本议定书微调的贡献。

1。第一天:病毒感染和缓冲区的制备<ol><li>准备5-10菜的组织培养细胞感染（直径15厘米），如PK - 15细胞为伪狂犬病病毒（PRV）Vero细胞或疱疹病毒（HSV）单纯。 </li><li>当细胞是95 - 100％融合，感染（MOI），5-10。要做到这一点，接种使用病毒库存总量在4毫升每盘每盘，然后孵育1小时板块在37 ° C。岩石板轻轻每15分钟一班，以确保细胞单层仍然完全由涂病毒接种。同时，热情为下一步的介质。 </li><li>感染一小时后，吸板从病毒接种，加入15毫升温介质，37 °培养箱在12-20小时的彗星。 </li><li>准备LCM的缓冲区（见表1）和岩石在4℃，充分搅拌过夜。病毒DNA再悬浮准备TNE（见表2）。 </li></ol> 2。第二天，第1阶段:细胞裂解和超速离心法<ol><li>目视确认感染的细胞造成统一的细胞病变效应（CPE）。允许感染的进展，直到所有的细胞都四舍五入，但没有关闭该板块解除。 </li><li>刮成中期感染的细胞，并组合成50 ml锥形管的细胞和介质。这一步所需的锥形管的数量将取决于感染步骤1.2以上菜的数量，锥形管例如，一个50毫升，可用于颗粒最大的3个菜 15 毫升培养基，每个可选:冲洗板用10 ml PBS，并结合细胞和介质。 </li><li>离心细胞和介质在2000年的相对centifugal力量在室温（RCF）10分钟，然后吸出上清液，并在每10毫升的PBS重悬沉淀。如果一个病毒感染的沉淀蔓延到多个步骤2.2锥形管，这些可重新组合成一个锥形管在这一步。重复离心和愿望可选:此颗粒可能被储存在-20 ° C和协议继续在以后的时间。 </li></ol> 3。第二天，第2阶段:超速离心 在以下的步骤，保持细胞沉淀和液晶显示模块缓冲区尽可能在冰上。 <ol><li> 0％甘油中添加β-巯基乙醇 - 液晶显示模块缓冲区。开始冷却超速离心机（以下步骤3.9），将它设置为4 ° C和转折点上的真空。 </li><li>细胞沉淀重悬在0％甘油5毫升 - LCM的缓冲区中，直到它不再是块状。如果有必要，涡管，打破任何不溶的细胞沉淀。 </li><li>提取物加入1.5毫升的氟里昂（1,1,2 - 三氯-2,2,2 - 三氟）和涡旋大力。立即在4 ° C的温度为10分钟2000 RCF离心收集与一个大口径的枪头（避免了接口）的顶层，并转移到一个新的管可选:如果颗粒是一种凝胶状固体，您可以很快地倒到一个新的管的顶层。 </li><li>重复提取氟利昂，离心一次。 </li><li>收集顶层（约5毫升），并转移到一个新的polyallomer超速离心机管。添加β-巯基乙醇，剩下的两个甘油 - 液晶显示模块缓冲区。 </li><li>准备渐变polyallomer离心管中，下部用3毫升，5％甘油氟利昂提取物（顶层） - LCM（中间层），然后再次下部用2.5毫升45％甘油 - LCM（底层）。使用薄移液器管的内容，以避免溢出。 </li><li>如果需要，准备等效平衡管LCM的缓冲区使用相同的比例。 </li><li>超速离心机桶转移到管。平衡水桶内彼此的0.1克，轻轻地添加额外的0％甘油 - 液晶显示模块缓冲区顶层（〜50-100一次μL）。 </li><li>使用水平或水平转头冷超速离心机，旋转平衡样品77000 RCF为SW41Ti转子（如25,000 RPM）在4 ° C的温度为1小时</li><li>在离心过程中，使玻璃挂钩浮动的DNA捕捉乙醇沉淀步骤（4.6以下）。巴斯德玻璃吸管的两端，并暂停对火焰的中间部分。当一段玻璃几乎熔化，轻轻拉伸展的玻璃，轻轻弯曲，创建一个紧密直角钩（&lt;90度），并大幅拉封锁的结束。如果玻璃钩提示是开放的，缓缴的火焰尖端，使玻璃融化，足以将其关闭。 </li><li>离心后，你应该看到一个在中间的暗斑薄不透明的颗粒。小心吸液管的，包括沿线两侧往，但避免沉淀在试管底部。 </li><li>在室温下加入0.5毫升TNE。 </li><li>让球团坐至少10分钟，让颗粒水化，Optional:颗粒也可能重悬在TNE过夜，如果保持在4 ° C。 </li></ol> 4。第三天:"人鬼情未了"的DNA沉淀<ol><li>分手用一个小口径的枪头（例如一个P200枪头）吹打的颗粒。不要担心在这一步，因为在这一点上衣壳中的病毒DNA仍然是剪切。添加下列试剂的试管中的病毒颗粒，在常温下，摧毁衣壳: <ul><li> 4.25毫升TNE </li><li> 0.25毫升10％SDS </li><li> 2毫克蛋白酶K </li></ul>轻轻摇动，混合使用这些试剂。应该有粘度略有增加。 </li></ol> 提防从这里剪切，如使用大口径的移液器和不旋涡病毒物质<ol start="2"><li>加入5毫升酚 - 氯仿提取病毒的蛋白质，并立即开始反转或震荡保持乳液。混合10秒钟，然后在室温下于2000 RCF离心5分钟的乳液。收集与一个大口径的枪头的顶层，避免了接口，并转移到一个新的管可选:使用锁相凝胶（PLG）管，在这一步，以避免污染从接口。 </li><li>重复的酚 - 氯仿抽提，离心一次。 </li><li>第二酚 - 氯仿提取后，收集到一个30毫升的玻璃Corex公司管的顶层和寒意管在-20 ° C为10-15分钟，照顾它不冻结。 </li><li>添加10毫升的冰冷乙醇，盖管（ 如拉伸封口膜中号的过顶的一块），反转立即混合。观察的DNA"鬼"出现，它由一个薄的粘稠沉淀。反转管轻轻拌匀进一步导致粘DNA沉淀集中在一起。 </li><li>使用玻璃钩捕获的DNA鬼（S）。仔细印迹的DNA，以除去多余的液体，然后放入1.5毫升管挂钩（尖向下），并晾干。添加0.25 - 0.5毫升的TE溶解DNA的鬼。允许再悬浮的DNA进行至少一小时。商店的DNA在4 ° C。 可选:为了最大限度地提高DNA产量，打破玻璃钩到1.5 ml管，使关闭的玻璃碎片再悬浮可以随着时间的推移继续。 </li></ol> 5。代表性的成果有了足够高MOI，细胞应达到充分的CPE〜18小时后感染（HPI）内为野生型伪狂犬病毒株，野生型HSV - 1株（图1）〜24 HPI。一旦被感染的细胞沉淀收获外，其余协议的步骤可以在一个漫长的一天完成或进行两天（图2）。 当准备玻璃钩子来捕获DNA的鬼，这是很重要的，使钩角小于90度，这样，以后它可以放入1.5毫升管基地（图3）。这个钩子的尖端封口防止吸管内的DNA损失。 DNA鬼形成过程中的沉淀步骤，并会显示为白色，粘稠的线程（图4）。这些聚合和坚持对方和/或用于捕获的DNA玻璃钩。因此，同样的玻璃挂钩可用于收集从多个线程的DNA内降水的解决方案。 <table border="1"><tbody><tr><td> 选择所需体积的列: </td><td> 10毫升 </td><td> 15毫升 </td><td> 20毫升 </td><td> 40毫升 </td></tr><tr><td> 1M KCL </td><td> 1.3毫升</td><td> 1.9毫升</td><td> 2.5毫升</td><td> 5毫升</td></tr><tr><td> 1M的Tris（pH值7.4） </td><td> 300μL </td><td> 450μL </td><td> 600μL </td><td> 1.2毫升</td></tr><tr><td> 1M氯化镁2 </td><td> 50μL </td><td> 75μL </td><td> 100μL </td><td> 200μL </td></tr><tr><td> 0.5 EDTA的</td><td> 10μL </td><td> 15μL </td><td> 20μL </td><td> 40μL </td></tr><tr><td> NP40/IGEPAL </td><td> 50μL </td><td> 75μL </td><td> 100μL </td><td> 200μL </td></tr><tr><td> β-巯基乙醇（添加使用前） </td><td> 4.3μL </td><td> 6.5μL </td><td> 8.6μL </td><td> 17.2μL </td></tr><tr><td colspan="5"></td></tr><tr><td colspan="5"> 使用所需％甘油（0，5，或45％）的比例</td></tr><tr><td> 0％的甘油</td><td>没有</td><td>没有</td><td>没有</td><td>没有</td></tr><tr><td>水</td><td> 8.3毫升</td><td> 12.5毫升</td><td> 16.7毫升</td><td> 33.4毫升</td></tr><tr><td> 5％的甘油</td><td> 0.5毫升</td><td> 0.8毫升</td><td> 1.0毫升</td><td> 2.0毫升</td></tr><tr><td>水</td><td> 7.8毫升</td><td> 11.7毫升</td><td> 15.7毫升</td><td> 31.4毫升</td></tr><tr><td> 45％Glycerol </td><td> 4.5毫升</td><td> 6.8毫升</td><td> 9.0毫升</td><td> 18.0毫升</td></tr><tr><td>水</td><td> 3.8毫升</td><td> 5.7毫升</td><td> 7.7毫升</td><td> 15.4毫升</td></tr></tbody></table> 表1。为LCM的缓冲区准备的食谱<table border="1"><tbody><tr><td> 对于50毫升的量</td></tr><tr><td> 5毫升1M氯化钠</td></tr><tr><td>三，pH值7.5 2.5毫升1M </td></tr><tr><td> 1毫升0.5 EDTA的</td></tr><tr><td> H 2 O 41.5毫升</td></tr></tbody></table> 表2。制备TNE（0.1 M氯化钠，50毫米的Tris，pH值7.5; 10毫米EDTA） <table border="1"><tbody><tr><td> 对于1升容积 </td></tr><tr><td> 0.2克氯化钾</td></tr><tr><td> 0.2克KH 2 PO 4 </td></tr><tr><td> 8 g氯化钠</td></tr><tr><td> 1.15克NA 2 HPO 4 </td></tr><tr><td> H 2 O至1L </td></tr></tbody></table> 表3。制备PBS（2.7毫米氯化钾1.5毫米KH 2 PO 4，137毫米氯化钠，8.1毫米的Na 2 HPO 4，pH值7.0） <img alt="图1" src="/files/ftp_upload/3151/3151fig1.jpg" /> 图1。PK15细胞（a）在感染，（二）中途感染，和（c）在细胞病变效应（CPE）后高的多重感染， 。 <img alt="图2" src="/files/ftp_upload/3151/3151fig2.jpg" /> 图2协议的概述。 <img alt="图3" src="/files/ftp_upload/3151/3151fig3.jpg" /> 图3。玻璃的三个有代表性的例子钩子用来收集病毒DNA鬼。 <img alt="图4" src="/files/ftp_upload/3151/3151fig4.jpg" /> 图4。良好的形成和丰富的"幽灵"的DNA有代表性的例子。

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没有利益冲突的声明。

本协议的某些部分最初是病毒DNA隔离BSL4条件，但它同样适应非BSL条件。4，我们通常使用此协议隔离α-疱疹病毒，伪狂犬病毒和HSV - 1，其中有DNA的基因组DNA封闭在一个蛋白质衣壳和脂质信封包围。7,8然而，它可能是直接适应其他大型DNA病毒，包括β-和γ-疱疹病毒和腺病毒。类似的提取通常用于RNA病毒以及9。 这个DNA的制备，净化多种方法，包括可能的?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>氟里昂（1,1,2 - 三氯-2,2,2 - 三氟） </td><td>费舍尔</td><td> T178 - 4 </td><td>请与您的机构适当处置的"指引"氟利昂含有废物，或者看到门德斯等。 9潜在的氟利昂替代品。 </td></tr><tr><td>锁相凝胶管，重型15毫升能力</td><td> 5 PRIME </td><td> 2302850 </td><td>可选</td></tr><tr><td> Polyallomer超速离心机管</td><td>贝克曼* </td><td> 331372 * </td><td> *选择管适合你自己的超速离心机，这些都是仅作为一个例子</td></tr><tr><td> NP - 40 / IGEPAL </td><td>西格玛</td><td>我- 3021 </td><td></td></tr><tr><td> PK - 15细胞</td><td> ATCC </td><td> CCL - 33 </td><td></td></tr><tr><td> Vero细胞</td><td> ATCC </td><td> CCL - 81 </td><td></td></tr><tr><td> PBS </td><td> Hyclone公司</td><td> SH30028.03 </td><td></td></tr></tbody></table>

preparation of viral dna from nucleocapsids

病毒是专性细胞寄生虫，因此它们的DNA的研究需要从宿主细胞中的污染物和DNA分离病毒的材料。几个下游应用需要大量的纯病毒DNA，这是由本议定书规定。这些应用程序包括病毒基因组测序，去除宿主DNA病毒序列数据输出是至关重要的优化，新的重组病毒株生产的，共转染纯化的质粒和线性病毒DNA有利于重组 。1,2， 3 此过程采用的提取和基于密度离心分离纯化线性疱疹病毒感染的细胞的核衣壳的DNA相结合 。4,5初步纯化步骤的目的是分离纯化病毒衣壳，其中包含和保护过程中的病毒DNA的提取和离心步骤去除细胞的蛋白质和DNA。核衣壳裂解，然后释放病毒DNA，最后两个酚 - 氯仿的步骤删除剩余的蛋白质。最后的DNA从溶液中捕获的是高度集中和纯粹，平均外径为 1.90 260/280。根据感染的细胞，病毒DNA范围从150-800微克或以上的产量的数量。这种DNA的纯度，使之稳定，在长期储存在4C。这种DNA，因此非常适合于高通量测序，高保真PCR反应，并转染。 开始前的协议，重要的是要知道每道菜的细胞（如平均15厘米在一个融合的菜，8 × 10 6 PK - 15细胞），和滴度的病毒的股票要使用的平均数（如1 × 10 8每毫升空斑形成单位）。这些是必要的适当的感染复数（MOI）计算的协议。， 例如 6，感染15 PK - 15细胞与上述病毒的股票在MOI为5厘米的菜，你会使用400μL病毒的股票和稀介质为3.6毫升（共接种4毫升，一个15厘米板卷）。 多个病毒DNA的制剂可以在同一时间准备。同时筹备工作的数量是有限的，只有超速离心机转子举行管的数量（每病毒之一;见下面的步骤3.9）。在这里，我们描述了，就像做一个病毒程序。

Watch this Scientific Journal Video about Preparation of Viral DNA from Nucleocapsids at JoVE.com

Preparation of Viral DNA from Nucleocapsids

我们描述了从被感染的细胞中分离高纯度的疱疹病毒衣壳DNA的过程。从溶液中捕获的是最后的DNA浓度和纯度高，使得它非常适合高通量测序，高保真PCR反应，并产生新的病毒重组体转染。

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. ...

preparation-of-viral-dna-from-nucleocapsids

Research

JoVE Journal

Immunology and Infection

20.8K 次观看.  Princeton University. 我们描述了从被感染的细胞中分离高纯度的疱疹病毒衣壳DNA的过程。从溶液中捕获的是最后的DNA浓度和纯度高，使得它非常适合高通量测序，高保真PCR反应，并产生新的病毒重组体转染。

视频: Preparation of Viral DNA from Nucleocapsids

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

质粒DNA重组流感病毒的一代

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

科研

免疫与感染

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

富达的RNA病毒和病毒的突变频率的表征的变种的分离

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

分离单链DNA，双链DNA和RNA病毒使用羟基磷灰石色谱从环境社区

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

一个简单的的Chelex协议对DNA的提取<em&gt;按蚊</em&gt;属。

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

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.

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

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

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

建立一个<em&gt;在体外</em&gt;系统来研究细胞内的行为<em&gt;光滑念珠菌</em&gt;在人THP-1巨噬细胞

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

使用Jsubtomo从电子Cryotomography重构平均病毒包膜糖蛋白穗

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

由腺病毒感染正常细胞分离病毒复制车厢富集亚核分数

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

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.

冻干环介导等温扩增试剂的检测中的发展<em&gt;贝氏柯克斯体</em

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