This protocol development was supported by the Bill and Melinda Gates Foundation Grand Challenges in Global Health grant 37774. Real-time PCR reagents and advice were provided by Abbott Molecular Inc. Des Plaines, IL. 8E5-LAV cells were provided by the Virology Quality Assurance Laboratory, Rush Presbyterian; St. Luke's Medical Center. HIV-1 negative blood was provided by Core Lab, NorthShore University HealthSystems, Evanston, IL. Thanks to Mark Fisher for photography help.

伦理声明:在本研究中使用的全血标本不被认为是涉及人类受试者的研究。用于临床诊断目的，这是满足了获得的样本，并为FINA研究测定提供这些试样的剩余部分。试样进行编码，使得研究者不能够容易地确定个人的身份。 1.国际泳联样品制备模块的研制<ol><li>准备用切割35×35毫米2.6 平方毫米厚的100％化妆棉吸水垫。切标本每一个焊盘。 </li><li>制备塑料石蜡膜与纸背衬带通过切割片石蜡膜（25毫米（H）乘50毫米（宽）），然后冲压在用锤子驱动打孔器磁带的中心的7.14毫米直径的孔。准备每个试样一个磁带。 </li><li>冲切8.35毫米直径的圆准备开往玻璃血液分离膜（捕获膜）盘用锤子驱动打孔。冲床每个试样一个磁盘。该盘具有2.3微米的颗粒截留能力。 </li><li>通过将捕获磁盘中使用镊子的印迹垫的中心组装FINA样品制备模块。放置在捕获磁盘石蜡膜带，使得石蜡带的孔离开露出的捕获盘的中心。 </li><li>通过稍微拉伸石蜡胶带以覆盖印迹垫并按下石蜡胶带牢固地将其粘到周围的磁盘的所有边缘的印迹垫确保磁盘和印迹垫之间的最大接触。 </li><li>作为实验所需或储存以备将来使用使尽可能多的模块。 </li></ol> 2.定量PCR管的制备<ol><li>制备5.1毫米磁带磁盘，将细胞捕获膜锚定在定量PCR管的一侧通过冲压双涂聚合α-的圆，以防止膜堵塞荧光检测STER诊断胶带从胶带的片材锤驱动冲头。准备每个试样一个磁带磁盘。 </li><li>移除胶带的贴衬垫的一侧。磁带放置到200μl的PCR管，粘着其上的一侧和朝向管的底部。删除第二个贴纸衬垫。管是现在准备好接收准备磁盘。 </li><li>作为实验所需或储存以备将来使用产生尽可能多的管子。 </li></ol> 3.图谋血液标本注意:如果正在准备一个真正的测试样品，继续步骤4。 <ol><li>解冻8E5-LAV细胞窝藏15从病毒学质量保证实验室获得的HIV-1前病毒的一个副本（VQA;拉什长老/圣路加医疗中心，芝加哥，IL）。 注意:它们被存储为在4000个细胞/μl浓度的冷冻细胞沉淀。如前面9中所述细胞计数进行了验证。 </li><li>准备SER胶质稀释在冷冻培养基（90％胎牛血清，10％二甲亚砜），以浓度范围从1至400个细胞/微升。 </li><li>吸管10微升细胞从各系列稀释成离心管中。添加100μl的新鲜的HIV-1阴性EDTA处理的全血样品的每个管来创建8E5-LAV拷贝数10-4,000的一个标准曲线。通过轻弹管的底部5次混合。 </li></ol> 4.执行国际泳联提取<ol><li>裂解血细胞中加入的Triton-X100），以1％（11微升10％的Triton-X100至110微升全血和来自步骤3.3的细胞）的最终浓度。 </li><li>通过轻弹管的底部5次混合。血应该把半透明的红色。 </li><li>吸取所有的血样到捕获盘。 注:石蜡带和捕获膜之间的水密封确保了血液流经膜，和捕获微米之间良好的接触embrane和印迹垫组件可确保快速样品排汗。 </li><li>允许样品通过过滤器浸泡。 注意:血液溶胞产物灯芯入印迹垫由于毛细管作用，捕捉俘获磁盘的表面上的基因组DNA。当它出现雾的裂解物已浸透到磁盘。 </li><li> 600-1000微升10毫米氢氧化钠逐滴加入到采集磁盘。 注意:洗涤缓冲液还可以加入作为本体加入600微升。缓冲器将形成疏水性石蜡的磁带上的微滴和通过在大约20秒的孔到盘芯吸。该过滤器会从红色变为血红蛋白的白色显示的间隙。 </li><li>分开用钳子印迹垫盘和盘适用于制备PCR管的胶带。如果有任何红色留在过滤器中添加50-100微升洗涤缓冲液在管放置之前清除过滤器。 注意:不经常，编写过程中施加过大压力从印迹垫分离期间在膜中的层的分区的FINA模块的结果。丢弃这些磁盘，并准备一个新鲜的样本。 </li><li>该过滤器被放置在PCR管后，分析样品的时候了。可替代地，在一个包含框硫酸钙干燥剂干燥过夜，然后盖和地点与二氧化硅干燥剂和存储的箔袋，直到准备用于分析。 </li></ol> 5.定量PCR反应<ol><li>使用HIV特异性引物和探针如前面所述9,11每准备100反应的qPCR微升主结构。包括内部放大控制并监控扩增抑制剂的存在和验证一个负样品是真阴性。确保过滤器完全覆盖主混合物和该过滤器保持固定到管的侧面整个反应。 </li><li>使用实时PCR装置如前面所述9执行放大。 </li></ol>

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The authors have nothing to disclose.

EID联系到快速治疗的访问已被证明是降低婴儿死亡率由于HIV感染16。由于婴儿的血液母源抗体的持久性，快速的艾滋病病毒抗体测试已确定HIV暴露婴儿的状态用处有限。世界卫生组织建议，出生HIV-1阳性母亲的婴儿都应该在4-6周龄用病毒学测试17进行测试。我们已经报道HIV-1的原病毒DNA的全血标本中检测和定量，能够单拷贝检测与证实100％的敏感性和特异性的61的中国婴儿11...

若干报告已经与使精美灵敏度和分子诊断的特异性可用于所有的目的讨论了点- -关心（POC）装置1-5的造纸或膜-基于提取方法中使用的开发。世界卫生组织（WHO）性传播疾病诊断倡议创造ASSURED（价格实惠，敏感，特异，用户界面​​友好，快速和强大，装备自由并交付给那些谁需要它）来描述理想的POC的特征词测试6。这些准则的免费装备，特点是特别具有挑战性，以实现分子诊断。然而，在该领域的每一次创新将推动实现这些最需要的目标，并有通过调整现有的技术7是希望短期内改善测试性能。 这里介绍一个简单的协议，用于从全血中提取DNA不需要复杂的化学或实验室仪器。国际泳联（核酸过滤隔离）的样品制备方法最初是提取全血白细胞DNA，以检测HIV-1前病毒定量PCR样品到答案点保健（POC）的一部分（定量PCR）婴儿早期诊断（EID）平台，在资源有限的设置8-11使用。 FINA提取不同于其中使用二氧化硅膜或氧化硅-包被的顺颗粒在离液剂12的存在下可逆地结合DNA常规纯化方法。相反，国际泳联采用立式过滤通过分离膜直接提取全血细胞DNA。含有包埋的DNA的膜可以直接在PCR管放置并且立即在购买扩增9干燥的PCR反应或空气中。没有离液剂，苯酚或醇是在样品提取使用，elimina婷去除提取过程13,14衍生有力的qPCR抑制剂需要大量洗涤步骤。 国际泳联膜可以捕获样品被添加到膜之前通过细胞裂解11解放或细胞9或基因组DNA。用于小区捕获，全血直接加入到该膜。细胞随后在膜中加入10毫摩尔的NaOH裂解。这种方法的优点是，它仅涉及3个步骤:1）加入样品; 2）细胞裂解/洗3）过滤盘放置在定量PCR管。这种方法的缺点是，该膜只能容纳一个定义单元成正比的膜盘的直径的数量。为了达到为EID所需的检测的限制，将100μl的全血需要这需要一个过滤器，过大，被放置在qPCR的管样品输入。裂解血细胞用洗涤剂添加样品到集合前膜增加了一个处理步骤，但允许对同一样本大小使用较小的滤波器。我们能够证明高再现性，单拷贝检测，并从100微升的血液使用这种测试配置11的HIV-1原病毒DNA的少至10个拷贝的定量。 在这份报告中，我们描述最初实验室使用开发的国际泳联协议。称为FINA样品制备模块的膜/滤膜夹层可以大批量制备，并储存起来以备后用。当试样被提取这个过程需要2分钟，并可以在不同大小的批次进行。在定量PCR可立即运行或包含嵌入DNA中的过滤器可以被存储直到它方便执行定量PCR。这种方法是用于在低和高资源设置标本常规分析非常方便。

<table><tbody><tr><td>Fusion 5</td><td>GE Healthcare Life Sciences</td><td>8151-9915</td><td></td></tr><tr><td>707 blotting pad</td><td>VWR International</td><td>28298-014</td><td></td></tr><tr><td>PARAFILM M</td><td>VWR International</td><td>52858-000</td><td></td></tr><tr><td>3M Double-Coated Polyester Diagnostic Tape&amp;nbsp;</td><td>3M Medical Specialties</td><td>9965</td><td></td></tr><tr><td>200 &amp;micro;l qPCR strip tubes</td><td>Agilent</td><td>401428</td><td></td></tr><tr><td>optical strip caps</td><td>Agilent</td><td>401425</td><td></td></tr><tr><td>Mx3005p qPCR System</td><td>Agilent</td><td>401456</td><td></td></tr><tr><td>sodium hydroxide</td><td>Sigma-Aldrich</td><td>221465</td><td>A.C.S. Reagent</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284</td><td>BioXtra</td></tr><tr><td>dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D8418</td><td>for molecular biology</td></tr><tr><td>fetal bovine serum, certified, U.S. origin</td><td>Thermo Fisher Scientific</td><td>16000-044</td><td></td></tr><tr><td>Hammer driven small hole punch 3/16&quot; hole diameter (5.1 mm)</td><td>McMaster Carr</td><td>3424A16</td><td></td></tr><tr><td>Hammer driven small hole punch 1/4&quot; hole diameter (7.14 mm)</td><td>McMaster Carr</td><td>3424A19</td><td></td></tr><tr><td>Hammer driven small hole punch 5/16&quot; hole diameter (8.35 mm)</td><td>McMaster Carr</td><td>3424A23</td><td></td></tr></tbody></table>

filtration isolation of nucleic acids:  a simple and rapid dna extraction method

FINA，核酸过滤分离，是通过分离膜和吸收垫利用垂直过滤提取从全血细胞的DNA，在不到2分钟的新颖的提取方法。血液检体与洗涤剂，短暂混合处理，并用移液管施加到分离膜。溶解物芯吸入印迹垫由于毛细管作用，捕捉分离膜的表面上的基因组DNA。期间的简单的洗涤步骤，其中PCR抑制剂是恶进入吸湿印迹垫提取的DNA被保留在膜上。然后将含有包埋的DNA的膜被加入到没有进一步纯化的PCR反应。这种简单的方法不需要实验室设备，并可以用廉价实验室用品很容易地实现。在这里，我们描述了从100微升全血中HIV-1前病毒DNA的高度敏感的检测和定量的协议作为一种模式在早期艾滋病毒的FANT诊断，可以容易地适用于其他遗传靶。

用于提取从8E5-LAV细胞掺入的全血前病毒DNA的工作流程示于图1， 图2示出了FINA示例模块和制备的qPCR管。这种方法允许从8E5-LAV细胞以不同拷贝数的HIV-1原病毒的有效扩增，如图做作标本（ 图3）的标准曲线。 PCR具有103％的效率，如从效率= 1 + 10（-1 /斜率）计算。线的方程为y = -3.25x + 27.95，与R 2 = 0.996的相关性。艾滋病毒原病毒DNA...

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

在这里，我们描述了一个简单，快速的基于纸张的DNA提取的HIV前病毒DNA的全血通过定量PCR检测方法。这种协议可以在检测其他遗传标记或使用替代的扩增方法被扩展为使用。

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 ...

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18.1K 次观看.  Northwestern University. 在这里，我们描述了一个简单，快速的基于纸张的DNA提取的HIV前病毒DNA的全血通过定量PCR检测方法。这种协议可以在检测其他遗传标记或使用替代的扩增方法被扩展为使用。 

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

Evaluating the Impact of Hydraulic Fracturing on Streams using Microbial Molecular Signatures

Hydraulic fracturing (HF), commonly called &#34;fracking&#34;, uses a mixture of high-pressure water, sand, and chemicals to fracture rocks, releasing oil and gas. This process revolutionized the U.S. energy industry, as it gives access to resources that were previously unobtainable and now produces two-thirds of the total natural gas in the United States. Although fracking has had a positive impact on the U.S. economy, several studies have highlighted its detrimental environmental effects. Of particular concern is the effect of fracking on headwater streams, which are especially important due to their disproportionately large impact on the health of the entire watershed. The bacteria within those streams can be used as indicators of stream health, as the bacteria present and their abundance in a disturbed stream would be expected to differ from those in an otherwise comparable but undisturbed stream. Therefore, this protocol aims to use the bacterial community to determine if streams have been impacted by fracking. To this end, sediment, and water samples, from streams near fracking (potentially impacted) and upstream or in a different watershed of fracking activity (unimpacted) must be collected. Those samples are then subjected to nucleic acid extraction, library preparation, and sequencing to investigate microbial community composition. Correlational analysis and machine learning models can subsequently be employed to identify which features are explanative of variation in the community, as well as identification of predictive biomarkers for fracking&#39;s impact. These methods can reveal a variety of differences in the microbial communities among headwater streams, based on the proximity to fracking, and serve as a foundation for future investigations on the environmental impact of fracking activities.

Here, we present a protocol to investigate the impacts of hydraulic fracturing on nearby streams by analyzing their water and sediment microbial communities.

使用微生物分子特征评估水力压裂对溪流的影响

Hydraulic fracturing (HF), commonly called &#34;fracking&#34;, uses a mixture of high-pressure water, sand, and chemicals to fracture rocks, releasing oil ...

科研

环境科学

DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been treated with storage/lysis buffer and archived at -80&deg;C, and to purify this DNA using a cesium chloride density gradient.  The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA.  Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract.  Part Five describes the optional purification via cesium chloride density gradient.  It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;m Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

从0.22微米Sterivex过滤器和氯化铯密度梯度离心法的DNA提取

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been ...

生物学

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

超高分子量基因组DNA的提取土壤和沉积物

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

快速分馏和全血的成分的分离样品中从社区为基础的设置获得

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

医学

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).

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 ...

免疫与感染

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

免电力，顺序的核酸和蛋白质的分离

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

使用洗涤剂和酶无酶方法从整个组织进行核分离

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

通过富集低 MW DNA 评估环境 AMR 的高效技术

Enhanced Extraction of Low-Molecular Weight DNA from Wastewater for Comprehensive Assessment of Antimicrobial Resistance

Environmental surveillance is recognized as an important tool for assessing public health in the post-pandemic era. Water, in particular wastewater, has emerged as the source of choice to sample pathogen burdens in the environment. Wastewater from open drains and community water treatment plants is a reservoir of both pathogens and antimicrobial resistance (AMR) genes, and frequently comes in contact with humans. While there are many methods of tracking AMR from water,&#160;isolating good-quality DNA at high yields from heterogeneous samples remains a challenge. To compensate, sample volumes often need to be high, creating practical constraints. Additionally, environmental DNA is frequently fragmented, and the sources of AMR (plasmids, phages, linear DNA) consist of low-molecular-weight DNA. Yet, few extraction processes have focused on methods for high-yield extraction of linear and low-molecular-weight DNA. Here, a simple method for high-yield linear DNA extraction from small volumes of wastewater using the precipitation properties of polyethylene glycol (PEG) is reported. This study makes a case for increasing overall DNA yields from water samples collected for metagenomic analyses by enriching the proportion of linear DNA. In addition, enhancing low-molecular-weight DNA overcomes the current problem of under-sampling environmental AMR due to a focus on high-molecular-weight and intracellular DNA. This method is expected to be particularly useful when extracellular DNA exists but at low concentrations, such as with effluents from treatment plants. It should also enhance the environmental sampling of AMR gene fragments that spread through horizontal gene transfer.

作者聚焦：通过将基于培养的技术和基因组学相结合来了解和检测环境抗菌素耐药性

Here, we present a simple technique to assess environmental antimicrobial resistance (AMR) by enhancing the proportion of low-molecular-weight extracellular DNA. Prior treatment with 20%-30% PEG and 1.2 M NaCl allows detection of both genomic and horizontally transferred AMR genes. The protocol lends itself to a kit-free process with additional optimization.

从废水中增强低分子量 DNA 的提取，用于抗菌素耐药性的综合评估 

Environmental surveillance is recognized as an important tool for assessing public health in the post-pandemic era. Water, in particular wastewater, has ...

核酸的分离过滤:一种简单快速的DNA提取方法

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此视频中的章节

使用微生物分子特征评估水力压裂对溪流的影响

从0.22微米Sterivex过滤器和氯化铯密度梯度离心法的DNA提取

超高分子量基因组DNA的提取土壤和沉积物

快速分馏和全血的成分的分离样品中从社区为基础的设置获得

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

免电力，顺序的核酸和蛋白质的分离

使用洗涤剂和酶无酶方法从整个组织进行核分离

从废水中增强低分子量 DNA 的提取，用于抗菌素耐药性的综合评估

从废水中增强低分子量 DNA 的提取，用于抗菌素耐药性的综合评估

从废水中增强低分子量 DNA 的提取，用于抗菌素耐药性的综合评估