The authors would like to acknowledge Latoya Wiggins and Katelyn Griffin for their assistance in sample acquisition, as well as Laura Lee Rutherford for their assistance in sampling and molecular analyses. We would also like to thank Sarah Owens from Argonne National Lab for microbiomic sample preparation and sequencing. These investigations were supported equally by the Agricultural Research Service, USDA CRIS Projects &#8220;Pathogen Reduction and Processing Parameters in Poultry Processing Systems&#8221; #6612-41420-017-00 and &#8220;Molecular Approaches for the Characterization of Foodborne Pathogens in Poultry&#8221; #6612-32000-059-00.

环境家禽生产样品1.机械同质化<ol style="margin-left: 40px;"><li>在提取之前，设定一水浴中以95℃，并让水浴时间达到该温度。 </li><li>称出0.33克土壤或粪便物质的到2ml裂解矩阵E管。 <ol><li>不超过0.33克在管样品的，因为这会导致以下的解决方案，以超过所述管的容量。 </li><li>解冻冷冻样品RT之前称重。 </li><li>为了共1克土壤/粪便进行分析，称出3复制0.33克样本为每个单独的环境样本。 </li><li>商店样品在萃取前-20℃圆锥形基质管内，如果需要的话。 </li></ol></li><li>添加825微升磷酸钠缓冲液和275微升的PLS溶液到样品管中。混合使用涡旋为〜15秒，然后离心样品在14000×g离心5分钟。 </li><li>倒出superna坦并加入700μlBuffer ASL的。混合使用涡旋5秒。 <ol><li>确保有顶部空间（〜10％总体积）在锥形管可用在这一点上。如果没有顶部空间，该管将有一种倾向在接下来的均化步骤可能导致交叉污染和/或样品损失泄漏。 </li></ol></li><li>将样品成FastPrep 24仪器，并且使样品均质化，在6.0米/秒，40秒的速度。 </li><li>离心匀浆样品在14000×g离心5分钟。将上清液转移到一个无菌的2ml微量管中。 </li><li>为了最大限度地提高来自样品的DNA回收率，重复步骤1.4至1.6，将上清液合并到同一无菌，加入2ml微量离心管中。 </li></ol>从样品匀浆抑制剂2.酶抑制注:此协议使用的QIAamp DNA凳子套件。 <ol style="margin-left: 40px;"><li>孵育上清液在95℃水浴中5分钟，以最大化从上清液内的任何剩余的细胞中的DNA的恢复。 <ol><li>孵育在70℃下对含有大部分革兰氏阴性生物体的样本。然而，如果革兰氏阳性生物体存在（这是与家禽粪便样品的情况下），孵育在95℃。 </li><li>使用在离心管塑料锁定夹以确保该管不会"弹出"开放式和可能丢失的样品体积作为压力可以在这些密封的离心管中建立起来的。 </li></ol></li><li>打开每个微量离心管中以释放压力，再盖的微量离心管，并混合使用涡旋15秒。 </li><li>离心样品以14000×g离心1分钟，取出1.2上清液毫升，并将其放置到一个新的无菌的2ml微量管中。 </li><li>加1 InhibitEx标签到每个样品中，并混合使用涡旋直到样品变得均匀白色/灰白色液体。 <ol><li>避免吨ouching所述InhibitEx标签而将其放入装有样品的离心管中。要做到这一点，将直接包含的标签在开放的离心管的泡罩包装，轻轻推离卡的泡罩包装，并移入离心管中。 </li></ol></li><li>孵育在室温（〜25℃）的样品1分钟，离心机在14000×g离心5分钟。 </li><li>所有的液体转移到一个无菌的1.5 ml离心管和离心机在14000×g离心5分钟。 <ol><li>避免传输液体时可能已沉淀在离心管的底部，在步骤2.5的末端任何剩余的颗粒。 </li></ol></li></ol>使用QIAcube工作站机器人工作站3.自动化DNA纯化注:塑料消耗品的数量，在离心机内的样本转子适配器的结构中，和缓冲器/溶液的所需体积依赖于NUMBER是正在运行的样品。 <ol style="margin-left: 40px;"><li>加入洗脱管和过滤管的转子适配器中的适当插槽。对于每个样品，加入400微升至转子适配器的中间槽。根据被纯化的样本数将转子适配器在工作站离心机在正确安排。 <ol><li>确保所有的离心管盖子被正确地固定在转子适配器中因为不这样做可能会导致在的离心步骤的纯化方案之一剪切。 </li></ol></li><li>加入1000微升和200微升过滤提示所需数量的工作站，并填写所提供的缓冲瓶缓冲所需音量。 注意:需要这种纯化方案（AL，AW1，AW2，和AE）的缓冲器都包含所述的QIAamp DNA粪便Mini试剂盒之内。用户需要提供所需要的AW BU的100％的乙醇ffers并且如在纯化过程中所用的溶液。 </li><li>添加提供的蛋白酶K的解决方案所需要的量进入无菌1.5 ml离心管，并将其放置到工作站上插槽A。此外，添加所需数量（等于样本的数量被净化）2毫升安全锁的离心样品管的RB到工作站的摇床截面。 <ol><li>保证样品管的盖子被安全地放置在工作站上的相应插槽中，因为不这样做将导致一个错误，当机器开始扫描工作站，以确保所有需要的塑料和液体可用于要求运行。 </li></ol></li><li>使用触摸屏的工作站上，选择DNA凳 - 人类凳 - 病原体检测协议，并通过后续屏幕阅读，以确保工作站被正确加载。一旦所有的检查屏幕被传递，选择开始运行这个协议。 <ol><li>如果从超过12个样品中提取DNA，开始均质化处理（步骤1）为下一个组样品中，由于12个样本的运行花费〜72分钟，以完成在工作站上。 </li></ol></li><li>从转子适配器在-20℃，直到需要用于后续下游分析取出样品，帽他们，和地点。 <ol><li>在这一点上，结合了3个重复纯化用于使用离心分离/蒸发为基础的系统的个体的样品（总分析量= 1克）。结合的重复和重新洗脱至100微升的Tris-ETDA缓冲器的最终体积。 </li></ol></li></ol>

<ol>
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The authors have nothing to disclose.

所使用的DNA提取方法影响的定量和定性的总细菌群落估计为粪便和垃圾样本，配套样品分析的DNA提取方法依赖性质1,3,6以前见过。对于这两种粪便和垃圾样本的DNA提取方法的性能订的是定量（机械&gt;混合&gt;酶）和质（酶&gt;混合&gt;机械）总细菌群落的估计不同。而混合方法没有产生最高定量或定性的估计，在两种情况下，混合方法产生的统计学相似性能最高的提取方法（ 图1，表2）...

When analyzing complex clinical or environmental samples (e.g., feces, soils), there are two main methodologies used for the extraction of DNA. The first is a mechanical disruption of the matrix using an intense bead-beating step, while the second is an enzymatic disruption of the matrix to chemically release bacterial cells and inhibit PCR inhibitors from the matrix simultaneously. Given the different means by which these two types of extraction methods work, it is not surprising that previous studies demonstrated that the appropriate DNA extraction method is both highly sample and analysis dependent. Comparative DNA extraction studies previously showed that some methods are more appropriate for improved DNA quality and quantity from environmental samples1-3, while others demonstrated improvements for community-level analyses such as denaturing gradient gel electrophoresis (DGGE)4-6, terminal restriction fragment length polymorphism (T-RFLP)7, automated ribosomal intergenic spacer analysis (ARISA)8, and phylogenetic microarrays9. Therefore, appropriate DNA extraction methods need to be used, or developed, according to the types of environmental samples and the types of analyses being performed on those samples, especially given the recent advancements in bacterial community analyses. 
Next generation sequencing, in conjunction with more quantitative community assessments (e.g., quantitative PCR (qPCR)), is becoming more prevalent in the environmental and clinical sciences, however, very little research has been performed to determine the effect of DNA extraction methods on these data sets. Most DNA extraction comparison studies dealt with microbiomic community estimates from human or human model samples10,11, not agricultural animal samples. The few poultry-focused next generation sequencing studies dealt with specific metagenomic12,13 or microbiomic14 questions; they did not discuss the effect of DNA extraction method on the resulting microbiomic analyses. Considering the complex nature of environmental samples related to poultry production (e.g., feces, litter/bedding, pasture soil), DNA extraction methods need to be carefully selected. Poultry-related environmental samples are known to contain large numbers of PCR inhibitors and up to 500-fold DNA extract dilutions have been required for PCR and subsequent downstream analysis15-17. Therefore it is essential that DNA extraction methods be optimized for these types of samples in order to not only physically disrupt the matrix, but also to be able to reduce/eliminate the large number of inhibitors that are present. 
The QIAamp DNA Stool Mini Kit, an enzymatic extraction method, has been considered the &#8220;gold standard&#8221; when extracting DNA from difficult gut/fecal samples1,18,19 and has been applied successfully to poultry environmental samples8,14. The enzymatic removal of PCR inhibitors through the use of a proprietary matrix is one of the greatest advantages of using this method for these types of environmental samples, as is the ability to significantly improve throughput (and reduce sample processing error) using automated workstations. One major disadvantage is the lack of a mechanical homogenization step to physically disassociate bacterial cells from the environmental matrix. When testing gut and fecal samples of non-poultry origin, the addition of a bead-beating or mechanical disruption step within a DNA extraction protocol significantly increased extraction efficiency9, DNA yield/quality1,4,5 and significantly improved downstream community analyses in terms of richness, diversity, and coverage5,6,11. These studies compared not only mechanical bead-beating methods to the &#8220;gold standard&#8221; enzymatic method, but some also added the mechanical bead-beating step to the enzymatic protocol to improve results6,9,11. 
According to the results from the above studies, bacterial community analyses (both qualitative and quantitative) could be improved from poultry-related environmental samples through the addition of a mechanical homogenization step to the enzymatic method. Therefore, the goal of this study was twofold: (1) to develop a novel DNA extraction technique that utilizes the most desirable aspects of both the mechanical (powerful homogenization step) and enzymatic (PCR inhibitor removal and automation) extraction methods and (2) compare the quantitative (via qPCR) and qualitative (via microbiomics) bacterial community assessments of this novel method to representative mechanical and enzymatic methods.

<table><tbody><tr><td>Lysing Matrix E tube</td><td>MPBio</td><td>6914-050</td><td>Different sizes available and the last 3 numbers of the cat. No. indicate size (-050 = 50 tubes, -200 = 200 tubes, -1000 = 1,000 tubes)</td></tr><tr><td>Sodium Phosphate Solution</td><td>MPBio</td><td>6570-205</td><td>Can be purchased individually, or also contained within the FastDNA Spin Kit for feces (Cat. No. 116570200)</td></tr><tr><td>PLS Buffer</td><td>MPBio</td><td>6570-201</td><td></td></tr><tr><td>Buffer ASL (560 ml)</td><td>Qiagen</td><td>19082</td><td></td></tr><tr><td>FastPrep 24 homogenizer</td><td>MPBio</td><td>116004500</td><td>48 x 2 ml HiPrep adapter (Cat. No. 116002527) available to double throughput of mechanical homogenization step</td></tr><tr><td>QIAamp DNA Stool Mini Kit</td><td>Qiagen</td><td>51504</td><td></td></tr><tr><td>QIAcube24 (110V)</td><td>Qiagen</td><td>9001292</td><td>Preliminary results show that QIAcube HT (Cat. No. 9001793) can be used to improve throughput, but different consumables are required of this machine and more comparative work needs to be done.</td></tr><tr><td>Filter-Tips, 1,000 ml (1024)</td><td>Qiagen</td><td>990352</td><td></td></tr><tr><td>Filter-Tips, 200 ml (1024)</td><td>Qiagen</td><td>990332</td><td></td></tr><tr><td>QIAcube Rotor Adapters (10 x 24)</td><td>Qiagen</td><td>990394</td><td>For 1.5 ml microcentrifuge tubes included with in the rotor adapter kit there is an alternative.&amp;nbsp; It is Sarstedt Micro tube 1.5 ml Safety Cap, Cat. No. 72.690</td></tr><tr><td>Sample Tubes RB (2 ml)</td><td>Qiagen</td><td>990381</td><td>Alternative: Eppendorf Safe-Lok micro test tube, Cat. No. 022363352</td></tr></tbody></table>

a hybrid dna extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples

的DNA提取协议的功效可以是高度依赖于样品被调查的的类型和执行的下游分析的类型。考虑到采用新的细菌群落分析技术（ 例如，microbiomics，宏基因组学）越来越流行在这些学科范围内的农业和环境科学等诸多环境样品可能是生理化学和微生物学的独特（ 如粪便和垫料/寝具样本家禽生产谱），适当和有效的DNA提取方法需要慎重选择。因此，一种新型的半自动混合DNA提取方法与环境家禽生产样品使用专门开发的。该方法是DNA提取的两种主要类型的组合:机械和酶。两步强烈的机械同质化的步骤（使用珠跳动专门针对环境下制定TAL样本）加入到"金标准"的酶的DNA提取方法的开始的粪便样品来增强去除细菌和DNA从样品基质和改善革兰氏阳性细菌的社区成员的恢复。一旦混合方法的酶法提取部分开始，剩下的净化过程是使用机器人工作站，以增加样品通量，降低样加工误差的自动化。在比较严格的机械和酶的DNA提取方法，这种新的混合方法加工的家禽粪便和垃圾样品时，考虑提供定量时（使用16S rRNA基因定量PCR）和定性（使用microbiomics）的总细菌群落的估计最佳的整体性能相结合。

在这项研究中，新鲜的粪便排泄物和垃圾样品从商业肉鸡舍（〜25000鸟）在美国东南部恢复。肉鸡（ 鸡内金 ）是柯布-500十字架，他们分别为59日龄，在采样的时间。新鲜的粪便和垃圾样本来自四个不同的区域恢复的房子内（靠近散热垫，附近的饮水器/馈线，在饮水器/馈线之间，靠近排风扇），并从这些地区的样本中含有5汇集样品从该区域内。从房子的四个方面的样本中提取定量/定性分?...

Watch this Scientific Journal Video about A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples at JoVE.com

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

一种混合DNA提取方法细菌群落的定性和定量评估，从家禽生产样品

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

a-hybrid-dna-extraction-method-for-qualitative-quantitative

Research

JoVE Journal

Biology

14.5K 次观看.  USDA-Agricultural Research Service. A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities. 

视频: A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

科研

遗传学

Hybrid De Novo Genome Assembly for the Generation of Complete Genomes of Urinary Bacteria using Short- and Long-read Sequencing Technologies

Complete genome sequences provide valuable data for the understanding of genetic diversity and unique colonization factors of urinary microbes. These data may include mobile genetic elements, such as plasmids and extrachromosomal phage, that contribute to the dissemination of antimicrobial resistance and further complicate treatment of urinary tract infection (UTI). In addition to providing fine resolution of genome structure, complete, closed genomes allow for the detailed comparative genomics and evolutionary analyses. The generation of complete genomes de novo has long been a challenging task due to limitations of available sequencing technology. Paired-end Next Generation Sequencing (NGS) produces high quality short reads often resulting in accurate but fragmented genome assemblies. On the contrary, Nanopore sequencing provides long reads of lower quality normally leading to error-prone complete assemblies. Such errors may hamper genome-wide association studies or provide misleading variant analysis results. Therefore, hybrid approaches combining both short and long reads have emerged as reliable methods to achieve highly accurate closed bacterial genomes. Reported herein is a comprehensive method for the culture of diverse urinary bacteria, species identification by 16S rRNA gene sequencing, extraction of genomic DNA (gDNA), and generation of short and long reads by NGS and Nanopore platforms, respectively. Additionally, this method describes a bioinformatic pipeline of quality control, assembly, and gene prediction algorithms for the generation of annotated complete genome sequences. Combination of bioinformatic tools enables the selection of high quality read data for hybrid genome assembly and downstream analysis. The streamlined approach for the hybrid de novo genome assembly described in this protocol may be adapted for the use in any culturable bacteria.

Hybrid De Novo Genome Assembly for the Generation of Complete Genomes of Urinary Bacteria using Short- and Long-read Sequencing Technologies

This protocol details a comprehensive approach for the culturing, sequencing, and de novo hybrid genome assembly of urinary bacteria. It provides a reproducible procedure for the generation of complete, circular genome sequences useful in studying both chromosomal and extrachromosomal genetic elements contributing to urinary colonization, pathogenesis, and antimicrobial resistance dissemination.

使用短读和长读测序技术生成尿细菌完整基因组的杂交从 头 基因组组装

Complete genome sequences provide valuable data for the understanding of genetic diversity and unique colonization factors of urinary microbes. These data ...

Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous catalytic enzymes and are naturally capable of metabolizing hydrocarbons. Scientists harness biodiversity in aquatic ecosystems to isolate bacteria with biodegradation and bioremediation potential. Such isolates from the environment provide a rich set of metabolic pathways and enzymes, which can be further utilized to scale up the degradation process at an industrial scale. In this article, we outline the general process of isolation, propagation, and identification of bacterial species from aquatic habitats and screen their ability to utilize hydrocarbons as the sole carbon source in vitro using simple techniques. The present protocol describes the isolation of various bacterial species and their subsequent identification using the 16S rRNA analysis. The protocol also presents steps for characterizing the hydrocarbon degrading potential of bacterial isolates. This protocol will be useful for researchers trying to isolate bacterial species from environmental habitats for their biotechnological applications.

We present the process of isolating, propagating, and characterizing hydrocarbon-degrading bacteria from aquatic habitats. The protocol outlines bacterial isolation, identification by the 16S rRNA method, and testing of their hydrocarbon-degrading potential. This article would help researchers in characterizing microbial biodiversity in environmental samples, and specifically screen for microbes with bioremediation potential.

从水生栖息地分离、繁殖和鉴定具有碳氢化合物代谢特性的细菌种类

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous ...

环境科学

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

超高分子量DNA提取微生物席

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

生物学

Production of Germ-Free Fast-Growing Broilers from a Commercial Line for Microbiota Studies

Studies of the gut microbiota contribution to the host physiology and immunocompetence are facilitated by the availability of germ-free animal models, which are considered the gold standard. Nesting birds are ideal models for the production of germ-free animals since there is no need to raise their relatives under sterile conditions. Germ-free chickens are mainly generated from specific-pathogen-free (SPF) experimental lines, which are poorly representative of commercial chicken lines.&#160;The method proposed here allowed the production of germ-free chickens from the fast growing broiler line Ross PM3, commonly used by the poultry industry.&#160;Eggs were quickly collected after laying at a broiler breeder farm.&#160;They underwent a strict decontamination process from the collection to the introduction in a sterile egg hatching isolator.&#160;The chicks have been hatched and kept in these sterile isolators during the period necessary to control their sterility. Originally developed for an experimental SPF white leghorn line, the present protocol has been adapted not only to the Ross PM3 broiler line but also to quails. It therefore represents a robust and readily adaptable procedure to other poultry species and nesting birds of economic, biological or ecological relevance.

Here we describe a method for the generation of germ-free chicks from eggs of a commercial broiler line, the Ross PM3. This method can be adapted for the generation of germ-free animals from other poultry species.

从用于微生物群研究的商业生产线上生产无菌快速生长的肉鸡

Studies of the gut microbiota contribution to the host physiology and immunocompetence are facilitated by the availability of germ-free animal models, ...

免疫与感染

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

高效的核酸提取和16S rRNA基因序列的细菌群落特征

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

从冷冻芯的外层部分外源物质的去除探讨古代生物群落里面窝藏

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These structures are collectively known as the nucleoskeleton and include the nuclear lamina, the nucleoli, and nuclear bodies. Although many variants of fluorescence in situ hybridization (FISH) exist to study the genome and its organization, these are often limited by resolution and provide insufficient information on the genome&#39;s association with nuclear structures. The DNA halo method uses high salt concentrations and nonionic detergents to generate DNA loops that remain anchored to structures within nuclei through attachment regions within the genome. Here, soluble nuclear proteins, such as histones, lipids, and DNA not tightly bound to the nuclear matrix, are extracted. This leads to the formation of a halo of unattached DNA surrounding a residual nucleus which itself contains DNA closely associated with internal nuclear structures and extraction-resistant proteins. These extended DNA strands enable increased resolution and can facilitate physical mapping. In combination with FISH, this method has the added advantage of studying genomic interactions with all the structures that the genome is anchored by. This technique, termed HALO-FISH, is highly versatile whereby DNA halos can be coupled with nucleic acid probes to reveal gene loci, whole chromosomes, alpha satellite, telomeres and even RNA. This technique provides an insight into nuclear organization and function in normal&#160;cells and in disease progression such as with cancer.

Combining DNA halo preparations with fluorescence in situ hybridization enables high-resolution analysis of genomic interactions with the nucleoskeleton. Attached genome leads to hybridized fluorescent signals located within the residual extracted nuclei, whereas non-attached genome is in the halo of DNA surrounding the residual nuclei.

DNA晕片制备物的荧光原位杂交揭示整个染色体、端粒和基因位点

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These ...

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic bacteria of special relevance for a wide number of hosts. P. syringae enters the plant from the leaf surface and multiplies rapidly within the apoplast, forming microcolonies that occupy the intercellular space. The constitutive expression of fluorescent proteins by the bacteria allows for visualization of the microcolonies and monitoring of the development of the infection at the microscopic level. Recent advances in single-cell analysis have revealed the large complexity reached by clonal isogenic bacterial populations. This complexity, referred to as phenotypic heterogeneity, is the consequence of cell-to-cell differences in gene expression (not linked to genetic differences) among the bacterial community. To analyze the expression of individual loci at the single-cell level, transcriptional fusions to fluorescent proteins have been widely used. Under stress conditions, such as those occurring during colonization of the plant apoplast, P. syringae differentiates into distinct subpopulations based on the heterogeneous expression of key virulence genes (i.e., the Hrp type III secretion system). However, single-cell analysis of any given P. syringae population recovered from plant tissue is challenging due to the cellular debris released during the mechanical disruption intrinsic to the inoculation and bacterial extraction processes. The present report details a method developed to monitor the expression of P. syringae genes of interest at the single-cell level during the colonization of Arabidopsis and bean plants. The preparation of the plants and the bacterial suspensions used for inoculation using a vacuum chamber are described. The recovery of endophytic bacteria from infected leaves by apoplastic fluid extraction is also explained here. Both the bacterial inoculation and bacterial extraction methods are empirically optimized to minimize plant and bacterial cell damage, resulting in bacterial preparations optimal for microscopy and flow cytometry analysis.

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

The present protocol describes a method that allows single-cell gene expression analysis on Pseudomonas syringae populations grown within the plant apoplast.

丁香假单胞菌基因在植物组织中表达的单细胞分析

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic ...

<meta charset="utf8"></head><body>鸡胃肠道中未培养微生物多样性的培养策略

Strategies to Enhance Cultivation of Anaerobic Bacteria from Gastrointestinal Tract of Chicken

The gastrointestinal tract (GIT) of chicken is a complex ecosystem harboring trillions of microbes that play a pivotal role in the host&#39;s physiology, digestion, nutrient absorption, immune system maturation, and prevention of pathogen intrusion. For optimal animal health and productivity, it is imperative to characterize these microorganisms and comprehend their role. While the GIT of poultry holds a reservoir of microorganisms with potential probiotic applications, most of the diversity remains unexplored. To enhance our understanding of uncultured microbial diversity, concerted efforts are required to bring these microorganisms into culture. Isolation and cultivation of GIT-colonizing microorganisms yield reproducible material, including cells, DNA, and metabolites, offering new insights into metabolic processes in the environment. Without cultivation, the role of these organisms in their natural settings remains unclear and limited to a descriptive level. Our objective is to implement cultivation strategies aimed at improving the isolation of a diverse range of anaerobic microbes from the chicken&#39;s GIT, leveraging multidisciplinary knowledge from animal physiology, animal nutrition, metagenomics, feed biochemistry, and modern cultivation strategies. Additionally, we aim to implement the use of proper practices for sampling, transportation, and media preparation, which are known to influence isolation success. Appropriate methodologies should ensure a consistent oxygen-free environment, optimal atmospheric conditions, appropriate host incubation temperature, and provision for specific nutritional requirements in alignment with their distinctive needs. By following these methodologies, cultivation will not only yield reproducible results for isolation but will also facilitate isolation procedures, thus fostering a comprehensive understanding of the intricate microbial ecosystem within the chicken&#39;s GIT.

<meta charset="utf8"></head><body>作者聚焦：推进家禽肠道细菌培养

The protocol presents two methodologies to improve the isolation of anaerobic intestinal bacteria. The first focuses on the isolation of a diverse range of bacteria using different culture media. The second focuses on the cultivation steps of a specific microbial group, possibly assimilating myo-inositol, to fully comprehend its ecological significance.

加强鸡胃肠道厌氧菌培养的策略

The gastrointestinal tract (GIT) of chicken is a complex ecosystem harboring trillions of microbes that play a pivotal role in the host&#39;s physiology, ...

一种混合DNA提取方法细菌群落的定性和定量评估，从家禽生产样品

摘要

探索更多视频

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

使用短读和长读测序技术生成尿细菌完整基因组的杂交从头基因组组装

从水生栖息地分离、繁殖和鉴定具有碳氢化合物代谢特性的细菌种类

超高分子量DNA提取微生物席

从用于微生物群研究的商业生产线上生产无菌快速生长的肉鸡

高效的核酸提取和16S rRNA基因序列的细菌群落特征

从冷冻芯的外层部分外源物质的去除探讨古代生物群落里面窝藏

DNA晕片制备物的荧光原位杂交揭示整个染色体、端粒和基因位点

丁香假单胞菌基因在植物组织中表达的单细胞分析

加强鸡胃肠道厌氧菌培养的策略

一种混合DNA提取方法细菌群落的定性和定量评估，从家禽生产样品

摘要

探索更多视频

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

使用短读和长读测序技术生成尿细菌完整基因组的杂交从 头 基因组组装

从水生栖息地分离、繁殖和鉴定具有碳氢化合物代谢特性的细菌种类

超高分子量DNA提取微生物席

从用于微生物群研究的商业生产线上生产无菌快速生长的肉鸡

高效的核酸提取和16S rRNA基因序列的细菌群落特征

从冷冻芯的外层部分外源物质的去除探讨古代生物群落里面窝藏

DNA晕片制备物的荧光原位杂交揭示整个染色体、端粒和基因位点

丁香假单胞菌基因在植物组织中表达的单细胞分析

加强鸡胃肠道厌氧菌培养的策略

使用短读和长读测序技术生成尿细菌完整基因组的杂交从头基因组组装