Name: a hybrid dna extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples
Uploaded: 2014-12-10T15:00:00
Description: 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

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?...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lysing Matrix E tube</td>
			<td style="width:71px;">MPBio</td>
			<td style="width:119px;">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 = 1000 tubes)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Phosphate Solution</td>
			<td style="width:71px;">MPBio</td>
			<td style="width:119px;">6570-205</td>
			<td>Can be purchased individually, or also contained within the FastDNA Spin Kit for Feces (Cat. No. 116570200)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PLS Buffer</td>
			<td style="width:71px;">MPBio</td>
			<td style="width:119px;">6570-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Buffer ASL (560 ml)</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">19082</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FastPrep 24 homogenizer</td>
			<td style="width:71px;">MPBio</td>
			<td align="right" style="width:119px;">116004500</td>
			<td>48 x 2 ml HiPrep adapter (Cat. No. 116002527) available to double throughput of mechanical homogenization step</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAamp DNA Stool Mini Kit</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">51504</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAcube24 (110V)</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">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 height="21">
			<td height="21" style="height:21px;width:216px;">Filter-Tips, 1000 ml (1024)</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">990352</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Filter-Tips, 200 ml (1024)</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">990332</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">QIAcube Rotor Adapters (10 x 24)</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">990394</td>
			<td>For 1.5 ml microcentrifuge tubes included with in the rotor adapter kit there is an alternative.&#160; It is Sarstedt Micro tube 1.5 ml Safety Cap, Cat. No. 72.690</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sample Tubes RB (2 ml)</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">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 ...

Research

JoVE Journal

Biology

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

使用Qiagen公司的小量制备试剂盒的细菌菌落纯化质粒DNA

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

科研

生物学

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

走在大鼠作为定性分析方法使用Rotorod

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

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

DNA Extraction from 0.22 ÃƒÅ½Ã‚Â¼M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

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 ...

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

一个外源凝集素的高效液相色谱法从复杂生物样品，以丰富的选择性糖基化肽

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

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 ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

从石蜡包埋材料的遗传和表观遗传分析的DNA提取

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Extraction of High Molecular Weight DNA from Microbial Mats

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 ...

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

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 ...

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

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 ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

混合式切割：用于顽拗植物组织样品的改进切法

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...