نود أن نشكر هيلتي أديسيتييو، دكتوراه، وشانجكسين يانغ، دكتوراه لتطوير البروتوكول. وقدمت الدعم الشامل للدولي الأمهات طب الأطفال المراهقين الإيدز السريرية المحاكمات المجموعة (إيمباكت) "المعهد الوطني للحساسية" والأمراض المعدية (نييد) من المعاهد الوطنية للصحة (NIH) ضمن "أرقام جائزة" UM1AI068632 (خط إيمباكت)، UM1AI068616 (إيمباكت سدمك) و UM1AI106716 (LC إيمباكت)، بتمويل مشترك من يونيس كينيدي شرايفر المعهد الوطني لصحة الطفل والتنمية البشرية (NICHD) والمعهد الوطني للصحة العقلية (NIMH). المحتوى هي المسؤولة الوحيدة عن المؤلفين ولا تمثل بالضرورة وجهات النظر الرسمية للمعاهد الوطنية للصحة.

<ol>
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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+placenta+samples+with+contamination+controls+does+not+provide+evidence+for+a+distinct+placenta+microbiota.">Comparison of placenta samples with contamination controls does not provide evidence for a distinct placenta microbiota.</a> Microbiome. 4 (1), 29 (2016).</li><li>Salter, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reagent+and+laboratory+contamination+can+critically+impact+sequence-based+microbiome+analyses.">Reagent and laboratory contamination can critically impact sequence-based microbiome analyses.</a> BMC Biol. 12, 87 (2014).</li><li>Walker, A. 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Mock microbial communities Available from: <a target="_blank" href="https://www.atcc.org/en/Products/Microbiome_Standards.aspx?utm_id=t170601524l1">https://www.atcc.org/en/Products/Microbiome_Standards.aspx?utm_id=t170601524l1</a> (2017)</li></ol>

الكتاب ليس لها علاقة بالكشف عن.

تستهدف الجيل التالي تسلسل الرنا الريباسي 16S تقنية تستخدم على نطاق واسع وسريع ل توصيف ميكروبيومي18. ومع ذلك، العديد من العوامل، بما في ذلك الآثار دفعة والتلوث البيئي، والتلوث عبر العينة، حساسية وإمكانية تكرار نتائج يمكن أن تضر بالنتائج التجريبية وارباك على تفسير7<...

يعتقد أن المجتمعات الميكروبية التي استعمار البشر أن تكون الأهمية لصحة الإنسان، والأمراض التي تؤثر على الأيض والتنمية محصنة والتعرض للمرض، والردود على التطعيم والمخدرات العلاج1، 2-التأكيد على الجهود المبذولة لفهم تأثير الحجمية على صحة الإنسان حاليا تحديد الميكروبات المرتبطة بالمكونات التشريحية المحددة (أي، الجلد، الأمعاء، عن طريق الفم، إلخ)، فضلا عن مواقع محلية داخل هذه المقصورات3،4. التي تدعم هذه الجهود التحقيق هو الظهور السريع، وزيادة إمكانية الحصول على تكنولوجيات الجيل التالي التسلسل (خ ع) التي توفر منصة موازية على نطاق واسع لتحليل محتوى الجينية الميكروبية (ميكروبيومي) من عينة. للعديد من عينات الفسيولوجية، ميكروبيومي المرتبطة بها على حد سواء معقدة ووفرة (أيالبراز)، ولكن لبعض العينات، ممثلة في ميكروبيومي بانخفاض الكتلة الحيوية الميكروبية (أي، واللبن البشري، والجهاز التنفسي السفلي) حيث حساسية والتحف الفنية التجريبية، والتلوث المحتمل تصبح القضايا الرئيسية. التحديات المشتركة في الدراسات ميكروبيومي والتصميم التجريبي المناسب قد خضعت متعددة استعراض المواد5،6،،من78.
المقدمة في هذه الوثيقة هو خط أنابيب تجريبية قوية خ ع استناداً إلى المستهدفة تسلسل الرنا الريباسي 16S V4 المنطقة9 تميز ميكروبيومي اللبن البشري. التحليل ميكروبيومي اللبن البشري معقدة ليس فقط طبيعتها منخفضة الكتلة الحيوية الميكروبية10، بل بالإضافة إلى ذلك بارتفاع مستويات الحمض النووي البشري الخلفية11،12،،من1314 و احتمال ترحيل PCR مثبطات15،16 في الحمض النووي المستخرج. يعتمد هذا البروتوكول على مجموعات استخراج المتاحة تجارياً ومنصات شبه الآلية التي يمكن أن تساعد في تقليل التغير عبر نموذج إعداد دفعات. ويضم مجتمع صورية بكتيرية المعالم التي يتم معالجتها جنبا إلى جنب مع عينات كعنصر تحكم الجودة للتحقق من صحة كل خطوة في البروتوكول، وتوفير مقياس مستقل من متانة الأنابيب. ورغم أن البروتوكول كوصف محدد لعينات اللبن البشري، وهو القابلة للتكييف وفقا لأنواع العينة الأخرى بما في ذلك البراز، المستقيم، المهبل، والجلد، ومسحات أريولار، وعن طريق الفم10،17، ويمكن أن تكون بمثابة نقطة انطلاق الباحثين الذين يرغبون في إجراء تحليلات ميكروبيومي.

<table><tbody><tr><td>AllPrep RNA/DNA Mini Kit</td><td>Qiagen</td><td>80204</td><td>DNA/RNA extraction kit</td></tr><tr><td>Eliminase</td><td>Fisher Scientific</td><td>435532</td><td>RNase, DNase, DNA decontaminant</td></tr><tr><td>Thermo Mixer</td><td>Fisher Scientific</td><td></td><td>temperature-controlled vortexer</td></tr><tr><td>Buffer RLT plus</td><td>Qiagen</td><td>1053393</td><td>guanidinium thiocyanate lysis buffer/ Part of Allprep kit</td></tr><tr><td>&amp;szlig;-Mercaptoethanol</td><td>Sigma Aldrich</td><td>63689-25ML-F</td><td>&amp;szlig;-ME is a reducing agent that will irreversibly denature RNases by reducing disulfide bonds</td></tr><tr><td>LME Beads</td><td>MP Biomedicals</td><td>116914050</td><td>bead tube</td></tr><tr><td>QIAgen TissueLyzer</td><td>Qiagen</td><td>85300</td><td>automated sample disruptor adapter set</td></tr><tr><td>QIAshredder column</td><td>Qiagen</td><td>79654</td><td></td></tr><tr><td>QIAgen RB tube</td><td></td><td></td><td>manufacturer&#039;s microcentrifuge tube in kit</td></tr><tr><td>QIAcube and related plasticware</td><td>Qiagen</td><td>9001292</td><td>automated DNA/RNA purification instrument</td></tr><tr><td>DNA exitus plus</td><td>Applichem</td><td>A7089</td><td>non-enzymatic decontamination solution</td></tr><tr><td>EB Buffer</td><td>Qiagen</td><td>19086</td><td>elution buffer</td></tr><tr><td>QIAgility and related plasticware</td><td>Qiagen</td><td>9001532</td><td>robotic liquid handler</td></tr><tr><td>PCR water</td><td>MO BIO</td><td>17000-</td><td></td></tr><tr><td>5PRIME HotMasterMix</td><td>Quantabio</td><td>2200400</td><td></td></tr><tr><td>Barcoded reverse primers</td><td>Eurofin</td><td>No Catalog #&#039;s</td><td>designed and ordered</td></tr><tr><td>96 well PCR plate</td><td>USA scientific</td><td>1402-9708</td><td></td></tr><tr><td>Tapestation 2200 and related plasticware</td><td>Agilent</td><td>G2964AA</td><td>automated DNA/RNA fragment analyzer</td></tr><tr><td>D1000 reagents for Tapestation</td><td>Agilent</td><td>5067-5585</td><td>Sample buffer and ladder are part of this kit</td></tr><tr><td>OneStep PCR Inhibitor Removal Kit</td><td>Zymo Research</td><td>50444470</td><td>PCR inhibitor removal is done per the manufacturer&#039;s instructions.</td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td>DNA clean up kit: silica-membrane-based purification of PCR products</td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Thermo Fisher</td><td>Q32854</td><td>dimethylsulfoxide-based dilution buffer and dye are part of this kit.</td></tr><tr><td>Qubit Fluorometer</td><td>Thermo Fisher</td><td>Q33216</td><td></td></tr><tr><td>NanoDrop</td><td>Thermo Fisher</td><td></td><td>microvolume spectrophotometer</td></tr><tr><td>MiSeq 300 V2 kit</td><td>Illumina</td><td>15033624/15033626</td><td></td></tr><tr><td>MiSeq</td><td>Illumina</td><td>No Catalog #&#039;s</td><td>next generation sequencer</td></tr></tbody></table>

a method for targeted 16s sequencing of human milk samples

وقد أصبحت دراسات المجتمعات الميكروبية على نطاق واسع مع وضع تسلسل غير مكلفة نسبيا والسريع، وارتفاع الإنتاجية. ومع ذلك، كما هو الحال مع جميع هذه التكنولوجيات، تعتمد النتائج استنساخه على سير عمل مختبر الذي يشتمل على الاحتياطات اللازمة والضوابط. وهذا أهمية خاصة مع العينات منخفضة-الكتلة الحيوية حيث تلويث الحمض النووي البكتيري يمكن أن تولد نتائج مضللة. هذه المادة تفاصيل سير شبه الآلي التعرف على الميكروبات من عينات حليب الثدي البشري استخدام تسلسل المستهدفة من منطقة V4 الريباسي الحمض النووي الريبي (الرنا الريباسي) 16S على نطاق throughput منخفضة إلى متوسطة. وصف البروتوكول إعداد عينة من الحليب الكامل الدسم بما في ذلك: عينة تحلل، واستخراج الحمض النووي، والتضخيم من منطقة V4 16S الرنا الريباسي الجينات، وإعداد مكتبة بتدابير مراقبة الجودة. الأهم من ذلك، البروتوكول والمناقشة النظر في القضايا التي البارزة في إعداد وتحليل العينات منخفضة-الكتلة الحيوية بما في ذلك الضوابط المناسبة الإيجابية والسلبية، وإزالة المانع بكر، تلوث العينة بالبيئية، الكاشف، أو مصادر تجريبية، وأفضل الممارسات التجريبية الرامية إلى ضمان إمكانية تكرار نتائج. حين البروتوكول كما هو موضح محددة لعينات اللبن البشري، أنها قابلة للتكيف للعديد من أنواع عينة الكتلة الأحيائية المنخفضة والعالية، بما في ذلك عينات جمعت في مسحات، مجمدة نظيفة، أو استقر في المخزن مؤقت الحفاظ على.

ويشمل البروتوكول المعروضة هنا خطوات مهمة مراقبة الجودة (مراقبة الجودة) لضمان أن تجتمع البيانات التي تم إنشاؤها بالنقاط المرجعية لبروتوكول التحكم حساسية وخصوصية، والتلوث. يتبع الأول QC خطوة البروتوكول [بكر] تضخيم المنطقة V4 16S (الشكل 2). ميليلتر واحد من المنت...

Watch this Scientific Journal Video about A Method for Targeted 16S Sequencing of Human Milk Samples at JoVE.com

A Method for Targeted 16S Sequencing of Human Milk Samples

ويرد سير شبه الآلي لتسلسل الرنا الريباسي 16S من اللبن البشري وأنواع العينة منخفض-الكتلة الأحيائية الأخرى المستهدفة.

أسلوب لتسلسل 16S المستهدفة من عينات اللبن البشري

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as ...

a-method-for-targeted-16s-sequencing-of-human-milk-samples

Research

JoVE Journal

Immunology and Infection

9.8K Views.  University of California at Los Angeles. ويرد سير شبه الآلي لتسلسل الرنا الريباسي 16S من اللبن البشري وأنواع العينة منخفض-الكتلة الأحيائية الأخرى المستهدفة.

Video: A Method for Targeted 16S Sequencing of Human Milk Samples

Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, optimization of their biotechnological synthesis is limited by the relatively low throughput of detection and quantification of monosaccharide and linkages. Conventional techniques of glycan analysis include chromatographic/mass-spectrometric methods with throughput on the order of hundreds of samples per day without automation. We demonstrate here, a genetically encoded bacterial biosensor for the high-throughput, linkage-specific detection and quantification of the fucosylated HMO structures, 2&#8217;-fucosyllactose and 3-fucosyllactose, which we achieved via heterologous expression of fucosidases. As the presence of lactose in milk or in biotechnological processes could lead to false positives, we also demonstrate the reduction of signal from lactose using different strategies. Due to the high throughput of this technique, many reaction conditions or bioreactor parameters could be assayed in parallel in a matter of hours, allowing for the optimization of HMO manufacturing.

We describe here the high-throughput detection and quantification of fucosylated human milk oligosaccharides (HMOs) using a whole-cell biosensor. We also demonstrate here, the adaptation of this platform towards analysis of HMO production strains, focusing on improving the signal to noise ratio.

تحليل تريساكتشاريديس الحليب فوكوسيلاتيد الإنسان في سياق التكنولوجيا الأحيائية باستخدام جينياً ترميز أجهزة استشعار العوامل البيولوجية

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, ...

Biochemistry

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

تحديد مسببات الأمراض البكتيرية النادرة التي 16S الريباسي جين تسلسل وMALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

الجيل المقبل من تسلسل خاص بالريبوسوم 16S RNA جين Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Biology

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

تنقية انجاس: تسلسل Metagenomes وMetatranscriptomes من مجمع عينات المرتبط الحيوانية

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and ...

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.

كفاءة نوكليك حمض استخراج و16S الريباسي جين تسلسل للبكتيريا الجماعة توصيف

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

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

وهناك طريقة لتقييم بكتريوسين الآثار على الأمعاء الدقيقة من الفئران

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. Deviations from what is considered a healthy microbiota composition (dysbiosis) may impair vital functions leading to pathologic conditions. Recent and ongoing research efforts have been directed toward the characterization of associations between microbial composition and human health and disease.
Advances in high-throughput sequencing technologies enable characterization of the gut microbial composition. These methods include 16S rRNA-amplicon sequencing and shotgun sequencing. 16S rRNA-amplicon sequencing is used to profile taxonomical composition, while shotgun sequencing provides additional information about gene predictions and functional annotation. An advantage in using a targeted sequencing method of the 16S rRNA gene variable region is its substantially lower cost compared to shotgun sequencing. Sequence differences in the 16S rRNA gene are used as a microbial fingerprint to identify and quantify different taxa within an individual sample.
Major international efforts have enlisted standards for 16S rRNA-amplicon sequencing. However, several studies report a common source of variation caused by batch effect. To minimize this effect, uniformed protocols for sample collection, processing, and sequencing must be implemented. This protocol proposes the integration of broadly used protocols starting from fecal sample collection to data analyses. This protocol includes a column-free, direct-PCR approach that enables simultaneous handling and DNA extraction of large numbers of fecal samples, along with PCR amplification of the V4 region. In addition, the protocol describes the analysis pipeline and provides a script using the latest version of QIIME (QIIME 2 version 2017.7.0 and DADA2). This step-by-step protocol is aimed to guide those interested in initiating the use of 16S rRNA-amplicon sequencing in a robust, reproductive, easy to use, detailed way.

This manuscript describes a detailed standardized protocol of high-throughput 16S rRNA-amplicon sequencing. The protocol introduces an integrated, uniformed, feasible, and inexpensive protocol starting from fecal sample collection through data analyses. This protocol enables analysis of large numbers of samples with rigorous standards and several controls.

بروتوكول المصحوبة بمرشدين لتوصيف الجراثيم البراز بتسلسل الرنا الريباسي-Amplicon 16S

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

تحليل شبه الجينومية السالمونيلا من غذاء والعينات البيئية

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

تحليل الكائنات المجهرية باستخدام ثنائي الخطوة PCR والجيل التالي من 16S rRNA التسلسل الجيني

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

<meta charset="utf8"></head><body>استعادة ميكروبيوتا الحليب المبستر المتبرع بها باستخدام حليب الأم

Individualized Reconstitution of Human Milk Microbiota: A Feasible Approach in Real-World Settings

Mother&#39;s own milk (MOM) is the most complete nutritional resource for newborns. In cases where mothers are unable to produce sufficient milk or cannot breastfeed, the preferred alternative is pasteurized donor human milk (PDM), which is routinely provided by human milk banks. PDM offers a superior range of nutritional and immunological elements compared to any commercially available formula. However, to ensure biosafety, PDM undergoes pasteurization, a process that inactivates commensal microbiota and reduces certain bioactive compounds. This study presents a protocol designed to restore the microbiota of PDM using MOM as a microbial source, adapting the approach to a real-world clinical setting.
The protocol was implemented in a clinical trial conducted at a maternity hospital and its associated human milk bank, with the aim of providing personalized donor milk to preterm infants whose mothers cannot produce sufficient milk. The methodology involves inoculating PDM with 10% of MOM, followed by incubation at 37 &#176;C for 4 h. Microbiological analysis demonstrated successful bacterial growth in the inoculated milk (IM) post incubation, with the microbiota profile of the reconstituted milk (RM) closely resembling that of MOM, indicating effective microbiota restoration. These results suggest that the reconstitution protocol is feasible for implementation in neonatal care, with the potential to enhance the nutritional and immunological quality of PDM, thereby supporting the health and development of non-breastfed newborns.

<meta charset="utf8"></head><body>إعادة التكوين الفردية لميكروبات الحليب البشري: نهج ممكن في بيئات العالم الحقيقي

The article describes a protocol for reconstituting pasteurized donor milk microbiota using the mother's own milk microbiota in practical, real-world settings. It demonstrates effective bacterial growth and microbiome modulation, supporting the feasible application of this procedure within the routine care of a maternity hospital and its associated human milk bank.

إعادة التكوين الفردية لميكروبات الحليب البشري: نهج ممكن في بيئات العالم الحقيقي

Mother&#39;s own milk (MOM) is the most complete nutritional resource for newborns. In cases where mothers are unable to produce sufficient milk or cannot ...