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Oral Microbiome Characterization in Murine Models

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Jan 2017



The oral microbiome has been implicated as a trigger for immune responsiveness in the oral cavity, particularly in the setting of the inflammatory disease periodontitis. The protocol presented here is aimed at characterizing the oral microbiome in murine models at steady state and during perturbations of immunity or physiology. Herein, we describe murine oral microbiome sampling procedures, processing of low biomass samples and subsequent microbiome characterization based on 16S rRNA gene sequencing.

Keywords: Oral microbiome (口腔微生物组), 16S rRNA gene sequencing (16S rRNA基因测序), Microbiome sequencing (微生物组测序), Murine oral microbiota (小鼠口腔微生物群), Gingival microbiome (牙龈微生物组), Mouse oral sampling (小鼠口腔取样)


The microbiome plays critical roles in modulating tissue-specific immune responses particularly at barrier sites (Belkaid and Harrison, 2017). In these barrier environments, such as the gastrointestinal tract and skin, select commensals are shown to be able to drive the development of specific immune cell populations (Ivanov et al., 2009; Naik et al., 2012). Our work has recently started to explore the influence of the oral microbiome in tailoring tissue immunity, particularly at the gingiva, a vulnerable oral barrier site (Abusleme and Moutsopoulos, 2016; Dutzan et al., 2017).

In humans, it is well recognized that the oral cavity harbors a diverse and rich microbiome (Human Microbiome Project, 2012). Alterations in oral microbial communities have been associated with the common oral disease, periodontitis, an inflammatory condition that affects the gingival tissues and results in tissue damage (Griffen et al., 2012; Abusleme et al., 2013; Moutsopoulos et al., 2015). To date, animal models have been instrumental in addressing the role of the microbiome in various physiological and pathological conditions (Turnbaugh et al., 2006; Kostic et al., 2013). However, studies of host-microbiome interactions have been increasingly challenging in the oral murine setting. To facilitate oral microbiome studies in murine models, we have developed protocols for sampling of oral microbial communities, processing of low biomass murine oral microbiome samples, 16S rRNA gene sequencing and analysis of relevant data.

Materials and Reagents

  1. Oral and gingival sample collection
    1. Ultra-Fine polystyrene swab (Puritan Medical Products, catalog number: 25-800 1PD 50 )
    2. Safe-lock Biopur 1.5 ml Individually wrapped tubes (Eppendorf, catalog number: 022600028 )
    3. KIMWIPES delicate task wipers (KCWW, Kimberly-Clark Professional, catalog number: 34120 )
    4. Sterile scalpel handle #3 (Fine Science Tools, catalog number: 10003-12 )
    5. Scalpel blades #10 (Fine Science Tools, catalog number: 10010-00 )
    6. 70% ethanol
    7. TE buffer (Epicentre, catalog number: MTE0970 )
    8. RNase AWAY decontamination reagent (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10328011 )
    9. Ketamine 100 mg/ml (Zetamine CIII, VetOne)
    10. Xylazine 20 mg/ml (AnaSed, Lloyd laboratories)

  2. DNA isolation
    1. Safe-lock Biopur 1.5 ml Individually wrapped tubes (Eppendorf, catalog number: 022600028 )
    2. DNA IQ spin baskets (Promega, catalog number: V1225 )
    3. DNeasy Blood and Tissue Kit (QIAGEN, catalog number: 69504 )
    4. Ready-Lyse lysozyme solution (Epicentre, catalog number: R1810M )
    5. Ethanol for molecular biology (Sigma-Aldrich, catalog number: E7023 )
    6. RNase AWAY decontamination reagent (Thermo Fisher Scientific, catalog number: 10328011 )

  3. 16S rRNA gene library amplification and visualization
    1. DNA low-bind tube 1.5 ml (Eppendorf, catalog number: 0030108051 )
    2. DNA low-bind tube 2.0 ml (Eppendorf, catalog number: 0030108078 )
    3. PCR tubes 0.2 ml without caps (Bio-Rad Laboratories, catalog number: TLS0801 )
    4. PCR tube 8-Cap strips (Bio-Rad Laboratories, catalog number: TCS0803 )
    5. Platinum Taq DNA polymerase high fidelity (Thermo Fisher Scientific, catalog number: 11304011 )
    6. dNTP mix (10 mM each) (Thermo Fisher Scientific, catalog number: R0191 )
    7. PCR water (QIAGEN, catalog number: 17000-10 )
    8. Forward primer 8F 5’-AGAGTTTGATCMTGGCTCAG-3’ (Custom order–IDT)*
    9. Reverse primer 361R 5’-CYIACTGCTGCCTCCCGTAG-3’ (Custom order–IDT)*
      *Note: These universal primers were first described in the article by Sundquist et al. (2007). Additionally, both forward and reverse primers need to include 5’ and 3’ linker sequences, heterogeneity spacers and the desired index identifier sequences that will allow dual indexing for later sample identification. These modifications are described in detail in the study of Fadrosh et al. (2014).
    10. UltraPure agarose (Thermo Fisher Scientific, catalog number: 16500500 )
    11. 50x TAE buffer (Bio-Rad Laboratories, catalog number: 1610743 )
    12. GelPilot DNA Loading dye, 5x (QIAGEN, catalog number: 239901 )
    13. UltraPure ethidium bromide (Thermo Fisher Scientific, catalog number: 15585011 )
    14. 100 bp DNA ladder (New England BioLabs, catalog number: N3231S )
    15. RNase AWAY decontamination reagent (Thermo Fisher Scientific, catalog number: 10328011 )

  4. PCR Product Clean-up, quantification and sequencing
    1. DNA low-bind tube 1.5 ml (Eppendorf, catalog number: 0030108051 )
    2. Qubit assay tubes (Thermo Fisher Scientific, catalog number: Q32856 )
    3. Agencourt AMPure XP (Beckman Coulter, catalog number: A63880 )
    4. Ethanol for molecular biology (Sigma-Aldrich, catalog number: E7023 )
    5. Buffer EB (QIAGEN, catalog number: 19086 )
    6. Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, catalog number: Q32851 )
    7. Experion DNA 1K Kit (Bio-Rad Laboratories, catalog number: 7007107 )
    8. PhiX Control V3 (Illumina, catalog number: FC-110-3001 )
    9. MiSeq Reagent Kit v3 (Illumina, catalog number: MS-102-3003 )
    10. RNase AWAY decontamination reagent (Thermo Fisher Scientific, catalog number: 10328011 )


  1. Sterile scissors (VWR, catalog number: 82027-582 )
  2. Sterile Fine scissors (Fine Science Tools, catalog number: 14058-11 )
  3. Sterile medium tipped forceps (Fisher Scientific, catalog number: 12-000-157 )
  4. Pipettes
  5. UV crosslinker (UV Stratalinker 1800) (Stratagene, model: Model 1800 )
  6. Microcentrifuge (Eppendorf, model: 5424 R )
  7. Heated shaking block for Eppendorf tubes (Eppendorf, model: Thermomixer 5436 )
  8. Vortex mixer (Scientific Industries, model: Vortex-Genie 2 )
  9. Spectrophotometer (Thermo Fisher Scientific, model: NanoDropTM 1000 , catalog number: ND-1000)
  10. Thermal Cycler (Bio-Rad Laboratories, model: DNA Engine )
  11. Electrophoresis Chamber (Bio-Rad Laboratories, model: Wide Mini-Sub Cell GT Cell )
  12. Gel Imager (Syngene, model: InGenius3 )
  13. DynaMag-2 Magnet (Thermo Fisher Scientific, model: DynaMagTM-2 Magnet )
  14. Automated electrophoresis station (Bio-Rad Laboratories, model: ExperionTM Automated Electrophoresis System )
  15. Experion priming station (Bio-Rad Laboratories, catalog number: 7007030 )
  16. Experion vortex station II (Bio-Rad Laboratories, catalog number: 7007043 )
  17. Qubit 3.0 Fluorometer (Thermo Fisher Scientific, model: Qubit 3.0 )


  1. Software Mothur (Schloss et al., 2009)
  2. Software R (R Core Team, 2017)
  3. Software RStudio (RStudio Team, 2015)


  1. Oral and gingival sample collection
    1. Oral mucosal microbiome sampling
      Note: This procedure allows for comprehensive evaluation of microbiome from all oral mucosal areas.
      1. Aseptically add 150 µl of TE buffer to 1.5 ml Safe-lock Biopur tubes. Include one per mouse to be sampled and one negative control.
      2. Clean work surface with 70% ethanol and RNase AWAY reagent. UV-treat tubes, racks, scissors for 30 min.
      3. Anesthetize or euthanize a mouse according to ARAC guidelines.
        Note: For anesthesia, we prepare a liquid solution consisting of Ketamine (100 mg/kg) and Xylazine (10 mg/kg), which is administered via intraperitoneal injection. We recommend that each mouse is weighed prior to injection, for dosing calculation.
      4. Wipe gloved hands with a tissue wipe previously moistened with RNase AWAY reagent.  
      5. Open swab envelope and have it ready (one hand), grab the mouse pulling the skin of the neck area so the mouth opens without touching it (other hand) (Figure 1A).
      6. Swab the oral cavity for 30 sec, starting on the tongue, then buccal areas, gingiva, palate and finish with the gingiva on the lower incisors (Figure 1B). 

        Figure 1. Oral swab sample collection. A. Hand positioning when handling the mouse for sampling, opening its mouth without touching it. B. Swab placement in the oral cavity, sampling tongue and buccal surfaces.

      7. Place the swab in TE buffer and cut the swab handle with scissors, to allow for the tube to close, but not leaving it too short. Place the tube immediately on dry ice. Collect a negative control swab, by taking a swab and exposing it to the air and then placing it into a tube with TE buffer.
      8. After collecting all samples, store them at -80 °C until processing.
    2. Gingival tissue sample collection
      Note: This procedure allows for evaluation of the oral microbiome at the gingiva and may be most useful in studies of periodontitis.
      1. Steps A2a-A2b as A1a-A1b.
      2. Euthanize a mouse according to ARAC guidelines.
        Note: For euthanasia, we expose the mice to CO2 in an appropriate chamber, following the guidelines established by the Institutional Animal Care and Use Committee, NIDCR/NIH.   
      3. Wipe gloved hands with a tissue wipe previously moistened with RNase AWAY reagent.  
      4. Perfuse the mouse and dissect tissues to expose the oral cavity (Figure 2A). These procedures are explained in detail in Dutzan et al. (2016).
      5. Using a #10 blade, dissect the palatal gingiva around the maxillary molars (Figure 2B). Remove gingival tissue with forceps and place it in a tube with TE buffer, which is directly transferred on dry ice. As a negative control, dip an unused pair of forceps in another tube with TE buffer.
      6. After collecting all samples, store them at -80 °C until processing. 

        Figure 2. Gingival tissue collection. A. Dissection of palatal gingival tissue around molar teeth from mouse maxilla. B. Enlarged view of the excised gingival tissue.

  2. DNA isolation
    1. Clean work surface with 70% ethanol and then RNase AWAY reagent. UV-treat a plastic tube rack, pipettes, sterile forceps and spin baskets (consider one pair of forceps and one basket per sample).
    2. Thaw previously collected samples by placing the tubes at 37 °C for 10 min on a heated shaking block at moderate speed.
    3. Briefly, spin sample tubes and add 1 µl of ReadyLyse per tube. Incubate for 60 min on a heated shaking block (37 °C) at moderate speed.
    4. Briefly spin tubes before opening.
    5. Add 25 µl of Proteinase K and 200 µl of buffer AL (both from the DNeasy Blood and Tissue Kit).
    6. Mix by vortexing briefly.
    7. Incubate overnight at 56 °C on a heated shaking block at a moderate speed.
    8. Next day, briefly spin tubes before opening.
    9. If samples are gingival tissues, continue to Step B12.
    10. If samples were collected with swabs, pull up the swab by grabbing it from the upper end with a pair of forceps, place a spin basket into the tube and now leave the swab into the basket.
    11. Take the tube with the basket and swab on top and proceed to briefly spin, to drain any liquid on the swab. Discard basket and swab.
    12. Add 200 µl of ethanol and mix well by vortexing.
    13. Briefly spin tubes before opening.
    14. Pipet the mixture into DNeasy mini spin column that was previously placed in a 2 ml collection tube (from now on all reagents are provided in the DNeasy Blood and Tissue Kit).
    15. Centrifuge at ≥ 6,000 x g for 1 min. Discard flow-through and collection tube.
    16. Place column in a new collection tube and add 500 µl of Buffer AW1.
    17. Centrifuge at ≥ 6,000 x g for 1 min. Discard flow-through and collection tube.
    18. Place column in a new collection tube and add 500 µl of Buffer AW2.
    19. Centrifuge at 20,000 x g for 3 min to dry column membrane. Discard flow-through and collection tube.
    20. Make sure column is dry before proceeding to next step, if it became wet, repeat Step B19 using a new collection tube.
    21. Place column in a new 1.5 ml Safe-lock Biopur tube (not included in the kit) and pipet 53 µl of Buffer AE directly onto the center of the membrane.
    22. Incubate for 5 min at room temperature.
    23. Centrifuge at ≥ 6,000 x g for 1 min.
    24. Measure DNA concentration in Spectrophotometer (Nanodrop, ND-1000).

  3. 16S rRNA gene amplification
    DNA concentration from oral murine samples (in health/steady state) is low, particularly when using the oral swab procedure (typically around 2-3 ng/µl of total DNA). Therefore, we modified our protocols for the subsequent PCR reactions adding 4 µl per reaction regardless of their DNA concentration. We also adjusted amplification conditions by increasing the number of PCR cycles (from 25 to 35) and decreasing the annealing temperature (from 52.5 °C to 50 °C). Importantly, our non-template controls and water alone did not amplify with this modified protocol. All PCR reactions per sample are performed in duplicate, then PCR products are pooled before purification.
    1. Clean work surface with 70% ethanol and then RNase AWAY reagent.
    2. Assemble master mix in a 2 ml LoBind tube, combine the required amounts of PCR water, dNTPs, MgSO4, Taq polymerase and buffer, according to the volume described below (per tube).

    3. Mix well by vortexing and add 13.6 µl of this mixture to each PCR tube.
    4. Add to each PCR tube the desired forward and reverse primer (both come with their own index sequence, for ‘dual indexing’).
    5. Add DNA template to each PCR tube.
    6. Place the tubes in a thermal cycler and run the following PCR conditions:

    7. Merge the duplicated PCR products from one sample in a DNA low-bind 1.5 ml tube. Then, run a 1.2% agarose gel using a 100 bp ladder, stain it with ethidium bromide or the DNA dye of your preference and visualize the PCR products in a gel imager. The expected amplicon size is ~528 bp.

  4. PCR product clean-up using the Agencourt AMPure XP system
    1. Clean work surface with 70% ethanol and then RNase AWAY reagent.
    2. Equilibrate AMPure XP Magnetic Particle Solution to room temperature (15-30 min).
    3. During that time prepare fresh 70% ethanol, aliquot the required amount of EB buffer (consider 40 µl per PCR product) and have ready a set of DNA low-bind 1.5 ml tubes.
    4. Measure the volume of each PCR product.
    5. Mix by vortexing the AMPure XP Magnetic Particle Solution, to re-suspend any magnetic particles that may have settled.
    6. According to the measured PCR product volume, add the appropriate amount of bead solution based on the following ratio (DNA:beads = 1:0.65).
    7. Vortex briefly and carefully, keeping the mixture in the bottom of the tubes. Incubate for 5 min at room temperature, for binding of DNA to beads.
    8. Place the tube onto a DynaMag-2 Magnet for 2 min or until beads separate from solution.
    9. While keeping the tubes in the magnet, carefully remove supernatant.
    10. Remove tubes from magnet and dispense 200 µl of 70% ethanol and pipet up and down to mix.
    11. Repeat Steps D8 and D9.
    12. Remove tubes from magnet and dispense 200 µl of 70% ethanol and pipet up and down to mix.
    13. Repeat Steps D8 and D9.
    14. While keeping the tubes in the magnet, open the tube lids and allow the beads to dry for approximately 5 min, making sure that the bead surface does not start to crack as this results in low DNA yield.
    15. Remove tubes from the magnet, add 40 µl of EB to each tube and mix well by pipetting.
    16. Place the tubes back in the magnet and wait for 2 min or until beads clearly separate from solution.
    17. Transfer ~35 µl of the supernatant to a new DNA low-bind 1.5 ml tube.

  5. PCR product quantification using the Qubit dsDNA HS Assay Kit
    1. Equilibrate kit solutions to room temperature (15-30 min).
    2. Set up the required number of Qubit assay tubes for samples and standards (2 tubes for standards).
    3. Label the tube lids.
    4. Prepare working solution by diluting HS reagent 1:200 in HS buffer. Prepare the desired amount considering that each sample will require 198 µl and the standards 190 µl.
    5. Add 190 µl of working solution to the standard tubes and add 10 µl of each standard to the appropriate tube. Mix by vortexing 2-3 sec, avoiding bubble formation.
    6. Add 198 µl of working solution to the sample tubes and add 2 µl of each sample to the appropriate tube. Mix by vortexing 2-3 sec, avoiding bubble formation.
    7. Incubate for 2 min at room temperature in the dark.
    8. Calibrate Qubit Fluorometer with the standards and proceed to read samples.
      Note: It is important to mention that the Qubit dsDNA HS Assay Kit detection range goes from 0.5 to 100 ng/µl. Samples below that detection range can be quantified, but their concentration may not be accurate.

  6. PCR product quantification and visualization using the Experion DNA 1K Kit
    We perform this step to confirm the purity of the PCR products after cleaning procedures. The size of the target DNA should correspond to a single peak at 528 bp.
    1. Equilibrate kit reagents to room temperature (15-30 min).
    2. While waiting, make sure that the DNA concentration of the PCR products doesn’t exceed 50 ng/µl, if it is above this value dilute to 50 ng/µl. For our samples, DNA concentration is usually below this value.
    3. Vortex briefly and short spin all reagents.
    4. Prepare gel-stain solution (GS) by adding 12.5 µl of stain (ST) to gel tube (G).
    5. Vortex for 10 sec and transfer the GS solution to a spin-filter tube (provided in the kit).
    6. Centrifuge spin-filter tube at 2,400 x g for 15 min.
    7. Discard filter. Date the tube as this GS solution lasts for 1 month. This solution needs to be protected from light and stored at 4 °C.
    8. Prime the DNA chip by adding 9 µl of GS to priming well, then select C3 on priming station and press Start. Visually inspect for bubbles or incomplete priming.
    9. Pipet 9 µl of GS to other GS wells (3) in the DNA chip.
    10. Pipet 5 µl of loading buffer into L well and each sample well (11).
    11. Pipet 1 µl of DNA 1K ladder into well L.
    12. Pipet 1 µl of DNA into each sample well. Add 1 µl TE buffer or DNase-free water in wells that were not used.
    13. Place chip in the Experion vortex station and press mix.
    14. Run the chip in the Experion electrophoresis station.

  7. PCR product pooling and sequencing
    1. Calculate the concentration in nM of each purified PCR product. Consider the amplicon size (528 bp) from Experion analyses and DNA concentration obtained from the clean PCR product using the Qubit.
    2. Dilute each PCR product to 4 nM using EB buffer. It is very important that PCR product concentrations and dilutions are accurate in order to obtain approximately equal number of reads per sample.
    3. Add 5 µl of each diluted PCR product to a 2.0 ml DNA low-bind tube, so samples are pooled in equimolar amounts. Measure library DNA concentration using Qubit assay as described before. Store at 4 °C.
    4. Pooled PCR products are ready to be sequenced on a MiSeq Instrument using the PhiX Control libraries and the MiSeq Reagent Kit v3 from Illumina, following the manufacturer’s standardized sequencing protocols.
    5. Prepare MiSeq reagents according to MiSeq® Reagent Kit v3 Reagent Preparation Guide.
    6. Considering DNA concentration measured in Step G4, proceed to denaturation and dilution steps described in MiSeq System Denature and Dilute Libraries Guide.
    7. For our samples, we diluted the library to 9 pM and combined it with 20 pM PhiX in a ratio that allows us to obtain a spike of PhiX of 14%.

Data analysis

  1. Following sequencing, the data is processed using the software Mothur (Schloss et al., 2009). A detailed description of the analysis pipeline is available in our original article (Dutzan et al., 2017). Briefly, the initial steps of pre-processing are aimed at eliminating low-quality reads, assembling contigs and filtering according to size (200-400 bp). Subsequently, we follow the MiSeq SOP pipeline (https://www.mothur.org/wiki/MiSeq_SOP) as described in Kozich et al. (2013). After pre-processing, sequences are clustered into Operational Taxonomic Units (OTUs) (using a 97% similarity cutoff). For taxonomic classification, we use the Ribosomal Database Project classifier (Wang et al., 2007) adapted for Mothur, which allows classification of OTUs up to genus-level. To improve OTU taxonomical identification, we then obtain the representative sequence for each OTU and compare it against the NCBI 16S rRNA database using BLAST. Top matches (presenting at least 97% similarity and coverage) provide additional species-level taxonomy information for each OTU.
  2. For data visualization, we use the software R and R studio, in conjunction with the R packages ‘ggplot2’ and ‘RColorBrewer’.
  3. To get an overview of the taxonomical composition of the samples, we typically plot the relative abundance for the top OTUs (mean abundance > 1%) (Figure 3). Differences in relative abundance can be determined using appropriate statistical tests (considering if data are paired, follow a normal distribution, etc.) and adjusting for multiple comparisons. We often use the LDA effect Size (LEfSe) tool (Segata et al., 2011) to identify differentially represented OTUs, which is available at https://huttenhower.sph.harvard.edu/galaxy/.

    Figure 3. Most abundant OTUs in oral microbial communities from gingival tissues and oral mucosal surfaces. Example data from 10-week-old C57BL6 mice (n = 10).

  4. To explore differences between communities (beta-diversity), samples can be compared using the ThetaYC distance, which measures dissimilarities in overall community structure. These data can be analyzed/visualized using Principal Coordinate Analysis (PCoA) (Figure 4).

    Figure 4. PCoA graph based on Theta YC distances. Samples from gingival and mucosal tissues cluster separately, indicating they harbor microbiomes with distinct global community structures (P < 0.001, determined by AMOVA).


Wear gloves, lab coat at all times and also consider wearing a mask when possible, to minimize risks of sample contamination during collection and processing.


This protocol does not require recipes for reagents, almost all reagents come ready to use.


The authors thank Dr. Nicolas Dutzan for assistance with photography and Ms. Teresa Wild for carefully reviewing this manuscript. This work was funded in part by the Intramural Program of the National Institute of Dental and Craniofacial Research (NIDCR) (to N.M.M), by grants R01 DE021578 and R21DE023967 (to P.I.D) from NIDCR/NIH, by the BBSRC (BB/M025977/1 to J.E.K) and by the Manchester Collaborative Centre for Inflammation Research (to J.E.K). This protocol was adapted from methods previously published in Dutzan et al. (2017). The authors do not have any conflict of interest or competing interests to declare.


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  20. Wang, Q., Garrity, G. M., Tiedje, J. M. and Cole, J. R. (2007). Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73(16): 5261-5267.


口腔微生物组被认为是口腔免疫应答的触发因素,特别是在炎性疾病牙周炎的形成中。 这里提出的协议旨在描述在稳定状态和扰动免疫或生理学的小鼠模型中的口腔微生物群。 在此,我们描述了鼠类口腔微生物群落取样程序,低生物量样品的处理和随后的基于16S rRNA基因测序的微生物群鉴定。

【背景】微生物组在调节组织特异性免疫应答(特别是在屏障部位)中起关键作用(Belkaid和Harrison,2017)。在这些屏障环境中,例如胃肠道和皮肤,选择的共生物显示能够驱动特定免疫细胞群体的发育(Ivanov等人,2009; Naik等人。,2012)。我们的工作最近开始探索口腔微生物组在剪裁组织免疫力方面的影响,尤其是在牙龈处,一个脆弱的口腔屏障部位(Abusleme和Moutsopoulos,2016; Dutzan等人,2017)。

在人类中,众所周知口腔中含有丰富多样的微生物(Human Microbiome Project,2012)。口腔微生物群落的改变与常见的口腔疾病,牙周炎(一种影响牙龈组织并导致组织损伤的炎症)有关(Griffen等人,2012; Abusleme等人。,2013; Moutsopoulos et al 。,2015)。迄今为止,动物模型已经有助于解决微生物组在各种生理和病理条件中的作用(Turnbaugh等人,2006; Kostic等人,2013) 。然而,宿主 - 微生物组相互作用的研究在口服鼠类环境中越来越具有挑战性。为了促进小鼠模型的口腔微生物组学研究,我们制定了口腔微生物群落取样,低生物量小鼠口腔微生物组样品处理,16S rRNA基因测序和相关数据分析的方案。

关键字:口腔微生物组, 16S rRNA基因测序, 微生物组测序, 小鼠口腔微生物群, 牙龈微生物组, 小鼠口腔取样


  1. 口腔和牙龈样本采集
    1. 超细聚苯乙烯拭子(Puritan Medical Products,目录号:25-800 1PD 50)
    2. 安全锁Biopur 1.5毫升单独包装管(Eppendorf,目录号:022600028)
    3. KIMWIPES微妙的任务刮水器(KCWW,金佰利专业,目录编号:34120)
    4. 无菌手术刀手柄#3(Fine Science Tools,目录号:10003-12)
    5. 手术刀刀片#10(精细科学工具,目录号:10010-00)
    6. 70%乙醇
    7. TE缓冲液(Epicentre,目录号:MTE0970)
    8. RNase AWAY去污试剂(Thermo Fisher Scientific,Invitrogen TM,产品目录号:10328011)
    9. 氯胺酮100毫克/毫升(Zetamine CIII,VetOne)
    10. 甲苯噻嗪20mg / ml(AnaSed,劳埃德实验室)

  2. DNA分离
    1. 安全锁Biopur 1.5毫升单独包装管(Eppendorf,目录号:022600028)
    2. DNA IQ旋转篮(Promega,目录号:V1225)
    3. DNeasy血液和组织试剂盒(QIAGEN,目录号:69504)
    4. Ready-Lyse溶菌酶溶液(Epicentre,目录号:R1810M)
    5. 乙醇分子生物学(西格玛奥德里奇,目录号:E7023)
    6. RNase AWAY去污试剂(Thermo Fisher Scientific,目录号:10328011)

  3. 16S rRNA基因文库的扩增和可视化
    1. DNA低结合管1.5毫升(Eppendorf,目录号:0030108051)
    2. DNA低结合管2.0 ml(Eppendorf,目录号:0030108078)
    3. PCR管0.2毫升无盖(Bio-Rad Laboratories,目录号:TLS0801)
    4. PCR管8-盖条(Bio-Rad Laboratories,目录号:TCS0803)
    5. 高保真Platinum Taq DNA聚合酶(Thermo Fisher Scientific,目录号:11304011)
    6. dNTP混合物(每种10mM)(Thermo Fisher Scientific,目录号:R0191)
    7. PCR水(QIAGEN,目录号:17000-10)
    8. 正向引物8F 5'-AGAGTTTGATCMTGGCTCAG-3'(Custom order-IDT)*
    9. 反向引物361R 5'-CYIACTGCTGCCTCCCGTAG-3'(Custom order-IDT)*
      注:这些通用引物首先在Sundquist等人的文章中描述(2007年)。此外,正向和反向引物都需要包括5'和3'接头序列,异质性间隔区和期望的索引标识符序列,这将允许双重索引以用于稍后的样品识别。在Fadrosh等人的研究中详细描述了这些修改。 (2014)。
    10. UltraPure琼脂糖(Thermo Fisher Scientific,目录号:16500500)
    11. 50x TAE缓冲液(Bio-Rad Laboratories,目录号:1610743)
    12. GelPilot DNA加载染料,5倍(QIAGEN,目录号:239901)
    13. UltraPure溴化乙锭(Thermo Fisher Scientific,目录号:15585011)

    14. 100 bp DNA梯(New England BioLabs,目录号:N3231S)
    15. RNase AWAY去污试剂(Thermo Fisher Scientific,目录号:10328011)

  4. PCR产物清洁,定量和测序
    1. DNA低结合管1.5毫升(Eppendorf,目录号:0030108051)
    2. Qubit测定管(Thermo Fisher Scientific,目录号:Q32856)
    3. Agencourt AMPure XP(Beckman Coulter,目录号:A63880)
    4. 乙醇分子生物学(西格玛奥德里奇,目录号:E7023)
    5. 缓冲液EB(QIAGEN,目录号:19086)
    6. Qubit dsDNA HS分析试剂盒(Thermo Fisher Scientific,目录编号:Q32851)
    7. Experion DNA 1K Kit(Bio-Rad Laboratories,目录号:7007107)
    8. PhiX Control V3(Illumina,目录号:FC-110-3001)
    9. MiSeq试剂盒v3(Illumina,目录号:MS-102-3003)
    10. RNase AWAY去污试剂(Thermo Fisher Scientific,目录号:10328011)


  1. 无菌剪刀(VWR,目录号:82027-582)
  2. 无菌精细剪刀(Fine Science Tools,目录号:14058-11)
  3. 无菌介质尖端钳(Fisher Scientific,目录号:12-000-157)
  4. 移液器
  5. UV交联剂(UV Stratalinker 1800)(Stratagene,型号:1800型)
  6. 微量离心机(Eppendorf,型号:5424 R)
  7. Eppendorf管加热摇动块(Eppendorf,型号:Thermomixer 5436)
  8. 涡旋混合器(Scientific Industries,型号:Vortex-Genie 2)
  9. 分光光度计(Thermo Fisher Scientific,型号:NanoDrop TM 1000,目录号:ND-1000)
  10. 热循环仪(Bio-Rad Laboratories,型号:DNA Engine)
  11. 电泳室(Bio-Rad Laboratories,型号:Wide Mini-Sub Cell GT Cell)
  12. 凝胶成像仪(Syngene,型号:InGenius3)
  13. DynaMag-2磁铁(Thermo Fisher Scientific,型号:DynaMag TM -2磁铁)
  14. 自动电泳站(Bio-Rad Laboratories,型号:ExperionTM自动电泳系统)
  15. Experion引发站(Bio-Rad Laboratories,目录号:7007030)
  16. Experion涡流站II(Bio-Rad Laboratories,目录号:7007043)
  17. Qubit 3.0荧光计(Thermo Fisher Scientific,型号:Qubit 3.0)


  1. 软件Mothur(Schloss 。,2009)
  2. 软件R(R核心团队,2017)
  3. 软件RStudio(RStudio团队,2015)


  1. 口腔和牙龈样本采集
    1. 口腔粘膜微生物群取样
      1. 无菌添加150微升的TE缓冲液1.5毫升安全锁Biopur管。
      2. 用70%乙醇和RNase AWAY试剂清洁工作表面。紫外线治疗管,架子,剪刀30分钟。

      3. 根据ARAC指南麻醉或安乐死鼠标 注:对于麻醉,我们制备一种由氯胺酮(100毫克/千克)和赛拉嗪(10毫克/千克)组成的液体溶液,通过腹腔注射给药。我们建议在注射之前对每只小鼠称重,进行剂量计算。
      4. 用先前用RNase AWAY试剂沾湿的纸巾擦拭戴手套的手。&nbsp;&nbsp;
      5. 打开棉签信封并准备好(单手),抓住鼠标拉动颈部皮肤,使嘴张开而不接触(另一只手)(图1A)。
      6. 擦拭口腔30秒,从舌头开始,然后颊部区域,牙龈,上颚和完成与下切牙牙龈(图1B)。

        图1.口腔拭子样本采集A.手柄定位时,处理鼠标进行采样,打开它的嘴而不接触它。 B.擦拭口腔,取样舌头和颊面。

      7. 将拭子置于TE缓冲液中,并用剪刀剪下棉签手柄,以使管子闭合,但不要太短。立即将管放在干冰上。收集阴性对照棉签,拭子并将其暴露于空气中,然后将其放入带TE缓冲液的管中。
      8. 收集所有样品后,将其储存在-80°C直到加工。
    2. 牙龈组织样本采集
      1. 步骤A2a-A2b作为A1a-A1b。
      2. 根据ARAC指南安乐死鼠标。
        注意:对于安乐死,我们将小鼠暴露于合适的室中的CO 2,遵循由Institutional Animal Care and Use Committee,NIDCR / NIH建立的指导方针。
      3. 用先前用RNase AWAY试剂沾湿的纸巾擦拭戴手套的手。&nbsp;&nbsp;
      4. 灌注老鼠并解剖组织以暴露口腔(图2A)。这些程序在Dutzan et al 中有详细的解释。 (2016)。
      5. 使用#10刀片,解剖上颌磨牙周围的腭牙龈(图2B)。用镊子取出牙龈组织,并将其置于带有TE缓冲液的管中,将其直接转移到干冰上。作为阴性对照,用TE缓冲液将一对未使用的镊子浸入另一个管中。
      6. 收集完所有样品后,将其储存在-80°C直到加工。

        图2.牙龈组织收集A.解剖来自小鼠上颌磨牙周围的腭齿龈组织。 B.切除牙龈组织的放大图。

  2. DNA分离
    1. 用70%乙醇清洗工作表面,然后用RNase AWAY试剂清洗。紫外线治疗塑料管架,移液器,无菌镊子和旋转篮(每个样品考虑一双镊子和一个篮子)。
    2. 解冻之前收集的样品,将试管置于37°C加热振荡块,中速10分钟。
    3. 简而言之,旋转样品管,每管添加1微升ReadyLyse。在中等速度的加热摇动块(37°C)上孵育60分钟。
    4. 在打开之前,简单地旋转管。
    5. 加入25μl蛋白酶K和200μl缓冲液AL(均来自DNeasy血液和组织试剂盒)。

    6. 在温度为56°C的高温摇床上以中等速度孵育
    7. 第二天,打开之前,简单地旋转管。
    8. 如果样品是牙龈组织,继续步骤B12。
    9. 如果用棉签收集样本,从上端用一把镊子抓住棉签,将一个旋转篮子放入管中,然后将棉签放入篮中。
    10. 拿着篮子的管子,在上面擦拭,然后短暂地旋转,以排出棉签上的液体。丢弃篮子和棉签。
    11. 加入200μl乙醇,涡旋混合。
    12. 在打开之前,简单地旋转管。
    13. 将混合物吸入先前置于2ml收集管中的DNeasy微型旋转柱(从现在开始,所有试剂均在DNeasy血液和组织试剂盒中提供)。
    14. 在≥6,000×g的条件下离心1分钟。丢弃流通和收集管。
    15. 将色谱柱置于新的收集管中,加入500μlBuffer AW1。
    16. 在≥6,000×g的条件下离心1分钟。丢弃流通和收集管。
    17. 将色谱柱置于新的收集管中,加入500μlBuffer AW2。
    18. 在20000×g离心3分钟以干燥柱膜。丢弃流通和收集管。
    19. 确保色谱柱已经干燥,然后进入下一步,如果它变湿了,使用新的收集管重复步骤B19。
    20. 放置一个新的1.5毫升安全锁Biopur管(不包括在工具包),并吸取53微升缓冲区AE直接到膜的中心。

    21. 在室温下孵育5分钟

    22. 在≥6,000×g的条件下离心1分钟
    23. 测量分光光度计(Nanodrop,ND-1000)中的DNA浓度。

  3. 16S rRNA基因扩增
    来自口服鼠样品(健康/稳定状态)的DNA浓度很低,特别是当使用口腔拭子程序(通常约2-3ng /μl总DNA)时。因此,我们修改了后续PCR反应的方案,无论其DNA浓度如何,每个反应添加4μl。我们还通过增加PCR循环次数(从25到35)和降低退火温度(从52.5℃到50℃)来调整扩增条件。重要的是,我们的非模板控制和水本身并没有放大与此修改的协议。每个样品的所有PCR反应一式两份进行,然后在纯化之前合并PCR产物。
    1. 用70%乙醇清洗工作表面,然后用RNase AWAY试剂清洗。
    2. 在2ml LoBind管中装配主混合物,根据下述体积(每个管)将所需量的PCR水,dNTP,MgSO 4,Taq聚合酶和缓冲液合并。

    3. 通过涡旋充分混合,并将13.6μl的该混合物加入到每个PCR管中。
    4. 为每个PCR管添加所需的正向和反向引物(两者都有自己的索引序列,用于“双重索引”)。
    5. 将DNA模板添加到每个PCR管。
    6. 将管置于热循环仪中并运行以下PCR条件:

    7. 将来自一个样品的重复PCR产物合并到一个DNA低结合的1.5ml试管中。然后,用100 bp的梯子进行1.2%的琼脂糖凝胶,用溴化乙锭或您喜欢的DNA染料染色,然后在凝胶成像仪上观察PCR产物。预期的扩增子大小约为528 bp。

  4. 使用Agencourt AMPure XP系统清洁PCR产物
    1. 用70%乙醇清洗工作表面,然后用RNase AWAY试剂清洗。
    2. 将Ampure XP磁性颗粒溶液平衡到室温(15-30分钟)。
    3. 在此期间,准备新鲜的70%乙醇,等分所需量的EB缓冲液(考虑每个PCR产物40μl),并准备好一套DNA低结合的1.5ml试管。
    4. 测量每种PCR产物的体积。
    5. 通过涡旋AMPure XP磁性颗粒溶液混合,重新悬浮任何可能沉降的磁性颗粒。
    6. 根据测量的PCR产物体积,根据以下比例(DNA:珠= 1:0.65)添加适量的珠溶液。
    7. 短暂地小心涡旋,将混合物保持在管的底部。
    8. 将管子放在DynaMag-2磁铁上2分钟,或直到珠子从溶液中分离出来。
    9. 在将管保持在磁铁中时,小心地移除上清液。
    10. 从磁铁上取下管子,分配200μl的70%乙醇,上下吸取混合。
    11. 重复步骤D8和D9。
    12. 从磁铁上取下管子,分配200μl的70%乙醇,上下吸取混合。
    13. 重复步骤D8和D9。
    14. 将试管保持在磁铁中,打开试管盖,让试管干燥约5分钟,确保试管表面不开裂,因为这会导致DNA产量低。
    15. 从磁铁上取下管,每管加入40μL的EB,并通过移液混合。
    16. 将管放回磁铁,等待2分钟,或直到珠粒清楚地从溶液中分离。
    17. 将约35μl的上清液转移至新的低结合量1.5ml管中。

  5. 使用Qubit dsDNA HS分析试剂盒进行PCR产物定量
    1. 将试剂盒溶液平衡至室温(15-30分钟)。
    2. 设置所需数量的样品和标准Qubit测定管(标准品2管)。
    3. 标注管盖。
    4. 通过在HS缓冲液中稀释HS试剂1:200来准备工作溶液。准备所需的数量,考虑到每个样品将需要198微升和标准190微升。
    5. 加入190微升的工作解决方案的标准管,并添加10微升的每个标准的适当管。通过涡旋2-3秒混合,避免气泡形成。
    6. 向样品管中加入198μl工作溶液,并将2μl的每个样品加入到相应的管中。通过涡旋2-3秒混合,避免气泡形成。

    7. 在室温下孵育2分钟
    8. 按照标准校准Qubit荧光计,然后继续阅读样品。
      注意:Qubit dsDNA HS检测试剂盒检测范围从0.5到100 ng /μl是很重要的。低于检测范围的样品可以量化,但其浓度可能不准确。

  6. 使用Experion DNA 1K Kit进行PCR产物定量和可视化
    1. 将试剂盒试剂平衡到室温(15-30分钟)。
    2. 在等待的过程中,确保PCR产物的DNA浓度不超过50 ng /μl,如果超过这个值,则稀释到50 ng /μl。对于我们的样本,DNA浓度通常低于这个值。
    3. 漩涡短暂旋转所有试剂。

    4. 在凝胶管(G)中加入12.5μl染色剂(ST),制备凝胶染色溶液(GS)
    5. 涡旋10秒钟,并将GS溶液转移到旋转滤管(在试剂盒中提供)。
    6. 将离心过滤管以2,400×g离心15分钟。
    7. 放弃过滤器。由于此GS解决方案持续1个月,所以请注明日期。这个解决方案需要避光保存在4°C。
    8. 通过添加9μL的GS灌注好的DNA芯片,然后在启动站选择C3,然后按启动。目测检查气泡或不完全启动。
    9. 吸取9微升GS到DNA芯片中的其他GS孔(3)。
    10. 吸取5μL上样缓冲液到L井和每个样品井(11)。
    11. 吸取1μL的DNA 1K梯子进入L型。
    12. 吸取1微升的DNA到每个样品井。
    13. 将芯片放在Experion涡流站中,然后按混合。
    14. 在Experion电泳站中运行芯片。

  7. PCR产物合并和测序
    1. 计算每种纯化的PCR产物的nM浓度。考虑来自Experion分析的扩增子大小(528bp)和使用Qubit从干净PCR产物获得的DNA浓度。
    2. 用EB缓冲液将每种PCR产物稀释至4nM。
    3. 每个稀释的PCR产物添加5μL到一个2.0毫升的DNA低绑定管,样品汇集在等摩尔量。如前所述使用Qubit测定法测量文库DNA浓度。在4°C储存。
    4. 按照制造商的标准化测序方案,使用PhiX控制文库和Illumina的MiSeq试剂盒v3,可以在MiSeq仪器上对合并的PCR产物进行测序。
    5. 根据 MiSeq ®试剂盒试剂准备指南
    6. 考虑到步骤G4中测量的DNA浓度,进行如 MiSeq系统变性和稀释库指南
    7. 对于我们的样品,我们将文库稀释到9pM,并将其与20pM PhiX以一定比例组合,使得我们能够获得14%的PhiX峰值。


  1. 在测序之后,使用软件Mothur处理数据(Schloss等人,2009)。分析流水线的详细描述可以在我们的原始文章(Dutzan et al 。,2017)中找到。简言之,预处理的初始步骤旨在消除低质量读取,按照大小(200-400bp)组装重叠群和过滤。随后,我们按照MiSeq SOP管道( https://www.mothur.org/wiki/MiSeq_SOP),如Kozich et al 中所述。 (2013年)。经过预处理后,序列被聚类到操作分类单元(OTU)(使用97%的相似性截止值)。对于分类学分类,我们使用适用于Mothur的核糖体数据库项目分类器(Wang et al。,2007),允许将OTUs分类到属级。为了改善OTU的分类鉴定,我们获得每个OTU的代表序列,并使用BLAST将其与NCBI 16S rRNA数据库进行比较。顶级匹配(呈现至少97%的相似性和覆盖率)为每个OTU提供额外的物种级分类信息。
  2. 对于数据可视化,我们使用软件R和R工作室,结合R包'ggplot2'和'RColorBrewer'。
  3. 为了对样品的分类组成进行概述,我们通常绘制出顶部OTU的相对丰度(平均丰度> 1%)(图3)。可以使用适当的统计测试(考虑数据是否配对,遵循正态分布,等等)来确定相对丰度的差异,并调整多重比较。我们经常使用LDA效应规模(LEfSe)工具(Segata et al 。,2011年)来识别差异代表的OTU,可在 https://huttenhower.sph.harvard.edu/galaxy/

    图3.来自牙龈组织和口腔粘膜表面的口腔微生物群体中的最丰富的OTU来自10周龄C57BL6小鼠的实例数据(n = 10)。

  4. 为了探索社区之间的差异(贝塔多样性),可以使用ThetaYC距离来比较样本,该距离衡量社区整体结构的差异。这些数据可以使用主坐标分析(PCoA)进行分析/可视化(图4)。

    图4.基于Theta YC距离的PCoA图表来自牙龈和粘膜组织的样品分别聚集,表明它们具有不同的全球群落结构的微生物组( P <0.001,测定由AMOVA)。






作者感谢Nicolas Dutzan博士和摄影师Teresa Wild女士仔细阅读本手稿。这项工作是由国家牙科和颅面研究所(NIDCR)(NMM)的校内项目资助的,由BIDRC(BB / M025977 / NIDCR)的NIDCR / NIH拨款R01 DE021578和R21DE023967 1到JEK)和曼彻斯特炎症研究协作中心(JEK)。该协议是根据以前在Dutzan等人发表的方法改编的。 (2017年)。作者没有任何利益冲突或竞争利益的申报。


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引用:Abusleme, L., Hong, B., Hoare, A., Konkel, J. E., Diaz, P. I. and Moutsopoulos, N. M. (2017). Oral Microbiome Characterization in Murine Models. Bio-protocol 7(24): e2655. DOI: 10.21769/BioProtoc.2655.