Name: identification of rare bacterial pathogens by 16s rrna gene sequencing and maldi-tof ms
Uploaded: 2016-07-11T13:00:00.000+00:00
Duration: 6 min 34 s
Description: Watch this Scientific Journal Video about Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS at JoVE.com

The authors would like to thank Prof. Enno Jacobs for his continuing support.

1.细菌DNA的提取<ol><li>的PBS溶液的制备<ol><li>称量1.65克Na 2 HPO 4×2H 2 O，0.22克的NaH 2 PO 4×2H 2 O 8.80克NaCl在一烧瓶中，并用蒸馏水补至1000 ml的终体积。将pH调节至7.4。对于最终使用过滤通过防菌（0.22微米）过滤该溶液。 </li></ol></li><li> DNA提取革兰氏阴性菌<ol><li>条纹在合适的培养基（ 如哥伦比亚血琼脂）病人资料，识别和隔离潜在的病原体。 </li><li>制备纯培养并以确定形态类型，并确认纯度和培养49执行革兰氏染色。 </li><li>选择一个单一的细菌菌落，并用1微升单用接种环将其传送到含有1毫升的PBS液无菌2.0毫升反应管中。 </li><li>孵育该悬浮液在一个THERMOMIX尔在95℃下进行10分钟。温育后，让细菌提取物（含有溶解的DNA），冷至室温，要么在-20直接将其用于扩增或商店℃下供进一步使用（ 图1a）。 </li></ol></li><li> DNA提取革兰氏阳性菌<ol><li>条纹在合适的培养基（如哥伦比亚血琼脂）病人资料，识别和隔离潜在的病原体。为了确认培养的形态型进行革兰氏染色。 </li><li>制备纯培养并以确定形态类型，并确认该培养的纯度进行革兰氏染色。 </li><li>选择一个单一的细菌菌落，用无菌1微升接种环，并在2.0毫升反应管中500微升PBS液将其挂起。 </li><li>加入500μl玻璃珠和管转移到一个振荡均化器，在最大的频率和幅度（50赫兹）操作时，5分钟。用1.0mm直径的玻璃珠破坏细菌的细胞壁。 </li><li>孵育该管在95℃10分钟并冷却至室温。使用该提取物可以直接用于在-20℃用于进一步使用PCR反应或存储。 </li></ol></li></ol> 2. 16S rDNA全PCR <ol><li>引物扩增的制备<ol><li>使用TPU1引物（5&#39;-AGA TGA GTT中医TGG CTC AG-3&#39;[M = A / C]）和作为反向引物正向引物和RTU4（5&#39;-TAC CAG GGT ATC TAA TCC TGT T-3&#39;） 25。制备的引物储备溶液用DNA酶和RNA酶的自由水，具有100皮摩尔/微升（100μM）的浓度，并进一步稀释至10皮摩尔/微升的工作溶液。存储底漆库存和工作溶液在-20℃或直接使用。 </li></ol></li><li> PCR反应混合物的制备<ol><li>吸管1μl的每种引物，1微升的dNTP混合（100毫摩尔），2.5微升的DNA提取物，5微升10×PCR缓冲液（含有25mM的MgCL 2），0.25微升的Taq聚合酶（5单位/微升）和39.25微升DNAse-和无RNA酶的PCR水进入无菌200微升反应管中。 </li></ol></li><li> PCR协议<ol><li>开始构建的PCR程序如下:首先，在95℃下的初始温育步骤持续5分钟，这是必要的，以激活的Taq聚合酶。第二，35个扩增循环，将含有1分钟。变性步骤在95℃，1分钟。引物退火步骤是在50℃，并在72℃1.5分钟的引物延伸步骤。第三，在72℃最终延伸10分钟。最后，将PCR反应物冷却至4℃。 注: 金黄色葡萄球菌和H 2 O作为阳性和阴性对照（ 图1b）。 </li><li>放置装有在热循环的PCR反应混合物中的管，并启动该程序。 </li></ol></li><li>的PCR产物的纯化 注:为了净化PCR产物，几个协议无论是可以使用基于酶或与二氧化硅吸附基质。这里使用的单步PCR清理利用两个水解酶反应。虽然核酸外切酶我（出一）消除单链DNA，虾碱性磷酸酶（SAP）水解未掺入的dNTP。第二个过程是基于用高盐快速结合的二氧化硅膜吸附技术 - 洗 - 低盐洗脱周期。 <ol><li>添加1.5微升含有外切我和SAP到25微升的PCR产物的酶混合物，调匀并在37℃，随后15分钟转移到施加前15分钟的简单的协议热循环仪在80℃。储存纯化的PCR产物或在-20℃或直接使用它作为目标标记的PCR。 </li></ol></li></ol> 3. DNA凝胶电泳<ol><li>电泳缓冲液（TBE缓冲液）的制备<ol><li>滴定54.0克（445毫摩尔）的TRIS碱，27.5mmol克（445毫摩尔）硼酸和20毫升的0.5M的EDTA（10毫摩尔）在一烧瓶中的溶液在pH 8.0与NaOH和用蒸馏水填充到1000毫升的总体积。然后用蒸馏水稀释（0.5×TBE）电泳此5×缓冲液1:10。 </li></ol></li><li>上样缓冲液的制备<ol><li>混合250毫克溴酚蓝，250毫克二甲苯蓝FF和15克多聚蔗糖（ 材料数据表 ）的入烧瓶中。然后用0.5×TBE缓冲液填充至100ml的总体积。分装样染料到1ml部分并储存在-20℃用于进一步的使用。 </li></ol></li><li>琼脂糖凝胶铸造<ol><li>称取6克标准琼脂糖电泳在烧瓶300毫升0.5X TBE缓冲液（参见3.1.1节），准备了2％（W ​​/ V）琼脂糖凝胶。然后把琼脂糖混合物在微波炉中，加热在接近沸点直到琼脂糖完全溶解，该溶液显示清晰。 </li><li>让熔融琼脂糖充分冷却，并添加1％10微升（重量/体积）溴化乙锭的库存SOLU化。将溶液倒入一个演员和放置中投凝胶梳子（允许30口井）。取出梳子当凝胶是完全凝固，把凝胶到含有0.5×TBE缓冲液的电泳室。 注:溴化乙锭是毒性和致突变。因此，应谨慎处理。最好是用丁腈手套。有可被用作无毒替代替代嵌入核酸污渍。 </li></ol></li><li> PCR产物的琼脂糖凝胶电泳 注意:为了验证对PCR扩增子的正确大小和来估计DNA浓度的纯化的PCR产物在琼脂糖凝胶上分离。 <ol><li>吸管2微升加样缓冲液的成PCR反应管中并加入8微升PCR产物。吸取这种混合到凝胶的孔中。在一个单独的孔加入8微升分子量大小标记（DNA梯）的估计PCR产物的大小。 </li><li>应用在10伏/厘米的恒定电压，并尽快停止电泳溴酚蓝标记已经到达凝胶的总长度的约¾。使用紫外透射（波长302纳米）可视化的分离的PCR片段并使用凝胶的文档系统记录它们。通过用该DNA梯子视觉比较估计的DNA的量。使用约10纳克目标为标记PCR。 </li></ol></li></ol> 4.桑格测序<ol><li>循环测序PCR 注:使用商业试剂盒（ 材料表 ）一个10微升反应执行PCR标记。 <ol><li>加入2微升5倍的反应反应体系的，2微升的DNA制备，1.5微升TPU1或RTU4工作液（10皮摩尔/微升）和4.5微升DNA酶和RNA酶的免费水。 </li><li>将这个测序反应混合物在热循环和运行其他PCR。总共，过程25个循环的变性步骤（96℃，10秒），退火步骤（45-60℃，5秒）和延伸步骤（60℃，2分钟）。最后，冷却反应至4℃。 </li></ol></li><li>测序反应的纯化<ol><li>净化使用商业离心柱的PCR纯化（ 材料表 ）标记反应混合物。 </li><li>负载10微升测序反应到预水合凝胶过滤基质中，并在750 xg离心执行离心步骤3分钟。 注:通过使用此程序非法人染料终止被保留在凝胶基质。 </li><li>然后干燥，在真空浓缩水洗脱液。离心机在2500×g离心20至30分钟，在40℃以完成干燥。 </li><li>添加10微升高度去离子的甲酰胺，然后在90℃下2分钟变性并在冰上冷却的混合物中。移取10微升的96孔微升板变性样品。在-20℃保存干燥，清洁的PCR产物如果分析在稍后的时间来进行。 </li></ol></li><li> 16S rRNA基因序列测定和DNA序列分析 注:在自动测序仪（ 材料表 ）进行序列分析。此处所用的定序器是一个自动化的基于荧光的毛细管电泳系统，同时分析4个样品（50毫升4-毛细管阵列）用液体聚合物67（ 图1c）。 <ol><li>放置在序微孔板。写在2奠定了样品表（板记录），保存它，并开始按照1给出的步骤的测序运行/分析。 注:序列运行时间为2小时，并提供了数据从800至1000基点。 </li><li>打开测序分析软件（ 材料表 ）。序列数据作为一个文本文件（.SEQ）和含有电泳包括质量值（QV）在一个文件（.ab1）。 注:基本通话将自动为每通过测序分析软件形成和底层电泳目视检查（ 例如 ，峰高，峰分离）的序列之前可用于进一步的应用。 </li><li>比较存储DNA序列文件的使用NCBI数据库NR BLAST算法（http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide）。 </li></ol></li></ol> 5. MALDI-TOF MS 注:质谱仪使用337纳米的 N 2激光和由特定的控制软件（ 材料表 ）操作。光谱被记录在线性模式和2,000〜20,000达覆盖之间的质量范围。数据解释和分配分数的样品被实时通过分析软件（ 材料数据表 ）（ 图1d）中进行。 <ol><li>细菌的制备MALDI-TOF MS分析<ol><li>增长的利益（ 例如 ，Myroides odoratimimus）上山口细菌含有5％马血，在37℃下进行18至24小时，这取决于细菌菌株（ 图2a）umbia血琼脂。执行革兰氏染色，以验证所研究的生物体的文化形态类型，以及纯度。 </li><li>使用木牙签为单个菌落转移至96-孔钢靶的孔中（ 图2b和2c）。点对钢靶在空气中干燥生物体的顶部1微升的70％甲酸和离开干燥约2-3分钟。 </li><li>然后，叠加与1μl的基质溶液的光斑，在有机溶剂中（50％乙腈，2.5％三氟乙酸，47.5％H 2 O）含50毫克/毫升的CHCA（α氰基-4-羟基肉桂酸）。 注意:酸覆盖方法（上板的制备方法）可被用于提高质谱的质量。已经显示，对于革兰氏阳性菌，例如一个"酸攻击"增加得分VA的测定光谱45的梅毒。 注:或者一个自动化样品制备系统（ 材料表 ）可以用来在其中一个第一干燥循环后1微升70％甲酸溶液，并自由接触斑点，1μl的基质溶液中的细菌涂片施加。然后将样品在60％相对湿度标准条件下干燥，并准备用于分析。 </li><li>让与基体的涂抹在引擎盖约5分钟覆盖风干一次。这与施加的矩阵细菌涂片是现在许多小时是稳定的。 注:无基质细菌涂片可能并不稳定，由于蛋白降解。 </li></ol></li><li>在进行MALDI-TOF MS分析<ol><li>样品简介<ol><li>打开我们的MMIS（ 材料表 ）的控制软件。 </li><li>通过按下仪器的IN / OUT按键通气质谱仪的样品装载端口。 </li><li>打开装货港点击后插入与干燥的细菌涂片加矩阵的MALDI-TOF MS目标板。 </li><li>关闭端口，并再次撤离。观察真空和当它达到4.5×10 -6毫巴开始测量。 </li></ol></li><li>样品分析<ol><li>打开我们的MMIS（ 材料表 ）的分析软件，然后点击"文件"菜单。选择"新建分类"和一个新窗口"MALDI biotyper实时分类向导"打开。键入项目名称，比如"Myroides测量PROJECT_1，在该领域"项目名称"，并按下按钮"新建"，在命名的新对话框中的"新建项目"检查项目名称，按下按钮进入"OK"。 </li><li>在"MALDI biotyper实时分类向导"观察"分析物的位置"窗口中打开。选择目标位置（ 例如 ，A1，A2，A3 ......），右键CLICK任何选定的位置（由一个蓝色的方块表示），并选择"添加样本"。在样品名称，样品编号和可选的注释自动打开表视图类型。点击"下一步"按钮继续。 </li><li>在"MALDI biotyper实时分类向导"守"MALDI选择biotyper方法"窗口中打开。选择"布鲁克分类学"，从"从分类树的MSP"，然后点击"下一步"继续。 </li><li>在"MALDI biotyper实时分类向导"观察"项目摘要"窗口中打开。检查上面给出实际的分类项目中插入条目。按按钮"完成"开始→运行的分类（ 图2d - F）。达到适当的真空时的测量自动启动。 </li><li>按照分类来看，直到测量顺利完成。查看在T结果表他"MALDI Biotyper实时分类项目Myroides测量PROJECT_1"打开菜单"查看"，然后单击"结果"。 </li><li>查看"布鲁克·道尔顿MALDI Biotyper分类结果"作为默认Web浏览器的HTML文件。打印并保存结果。 </li></ol></li><li>测量试验标准日之前，样品分析校准仪器并执行仪器验证每周（仪器自检）。 注:在测量MALDI-TOF光谱的分析软件（ 材料数据表 ）实时进行了分析。十个品种各自的成绩列表，并给出送入汇报了实验室信息系统（LIS）。 </li><li>严格评估MALDI-TOF MS的结果（见代表性的结果），并且如果合适的话，包括其他测试，例如biochemical-及药敏型材或16S rRNA基因测序和，如果需要的话，革兰氏染色分配细菌种类的微生物报告。 </li></ol></li></ol>

The authors declare that they have no competing financial interests.

两个MALDI-TOF MS和16S rRNA基因测序提供识别大量不同的细菌的可能性。 MALDI-TOF MS是一个快速和廉价的方法，这是易于处理和细菌质谱大型数据库是可用的。出于这个原因，MALDI-TOF MS是进行集中在罕见的细菌病原体17,20,39,51筛选研究一种快速，成本有效和可靠的方法。在前瞻性研究中比较MALDI-TOF MS和其他表型的鉴定方法，诚等人证明成本效益和MALDI-TOF MS 59和Tan的速度等人?...

在常规诊断罕见病原体安全识别是由以下事实古典文化和生物化学方法是繁琐，有时可疑阻碍。此外，诊断微生物学实验室必须处理大量的病原体，范围从几百到几千，每天，这需要使用自动化系统。除了高每日吞吐量的管理，需要细菌物种的精确识别。因为他们在抗菌药物敏感性的模式不同，因此正确识别提供了选择适当的抗生素的基本信息（ 如， 肠球菌 ， 不动杆菌属）12,43医生这是必要的。 全自动微生物鉴定系统（AMIS）应用规范组酶促反应的表征细菌分离株的代谢特性<sup&gt; 13,15,16,26,27。虽然在这些系统中使用的墨盒利用大量不同的生化反应的， 例如，47在本研究中52所用的AMI的GN卡，这种策略允许安全识别只为一组有限的细菌。此外，数据库，一个先进的专家系统，显然是集中在检测的医学重要性13,15,16,36相关的和高度相关的细菌。另外两个系统，广泛应用于实验室，也适用于细菌鉴定这种生化途径。最近的研究表明在该研究中使用的的AMI和竞争对手之一（93.7％和93.0％）之间的比较的识别精度，同时第三阿美族具有只有82.4％的识别精度上物种水平35。这种差异可能由底层识别数据的引用，包和软件，在麦太保差异的版本的质量来解释栓塞技术人员的35,36精通。 两条自动化MALDI-TOF MS系统（MALDI-TOF微生物鉴定系统，MMIS）主要使用。这些系统允许检测大量基于其蛋​​白质指纹质谱细菌种类的。例如，所用的MMIS的数据库包含6000参考光谱。基于质谱鉴定系统提供的种类繁多的微生物，包括致病菌罕见的快速11,48,51和可靠的检测。迄今只有少数直接比较是在本研究中使用的MMIS和其竞争者19,33之间提供。根据Daek 等人的这两个系统提供识别精度的类似的高率，但是在本研究中使用的MMIS似乎是在物种鉴定19更可靠。 同样，分子技术寻址非常保守，而且不同的基因（ <em&gt;如16S rDNA全或的rpoB）允许一个明确的物种鉴定3,22,61。这其中，16S rDNA的是因为它在所有的细菌34存在的最广泛使用的持家基因。其功能保持不变，并且最后，用大致1500 bp的，这是足够长，以适合于生物信息14,34。许多研究者认为16S rRNA基因分析的"黄金标准"进行细菌鉴定21。这是由于这样的事实，即几个实验室使用DNA-DNA杂交技术迄今为罕见的或新的细菌14,34的鉴定。此外，越来越多的数据库可用，可用于16S rRNA基因分析50。但是，它必须考虑到，基于16S rDNA的检测系统相比，标准PCR方案有一个有限的敏感度。此外，该分子的方法是复杂的，耗时的，并且需要训练有素的人员，以及专门的实验室设施的，因此，不容易落实到日常诊断55。此外，已经表明，细菌鉴定至少两种不同的方法的组合导致高度精确的菌种鉴定。 MALDI-TOF MS和16S rDNA序列的结合允许大量以高精度不同细菌种类的识别。最近提出进行细菌鉴定流行病学研究的问题和罕见的病原体56 MALDI-TOF MS和16S rRNA基因分析的结合。

<table><tbody><tr><td>CHROMASOLV, HPLC grade water, 1 L</td><td>Sigma-Aldrich Chemie, M&amp;uuml;nchen, Germany</td><td>270733</td><td></td></tr><tr><td>Tissue Lyser LT</td><td>Qiagen, Hilden, Germany</td><td>85600</td><td>Oscillating homogenizer</td></tr><tr><td>Glass-beads 1.0 mm</td><td>VWR International, Darmstadt, Germany</td><td>412-2917</td><td></td></tr><tr><td>Thermomixer 5436</td><td>Eppendorf, Hamburg, Germany</td><td>2050-100-05</td><td></td></tr><tr><td>QIAamp DNA Mini Kit (250)</td><td>Qiagen, Hilden, Germany</td><td>51306</td><td></td></tr><tr><td>Taq PCR Core Kit (1,000 U)</td><td>Qiagen, Hilden, Germany</td><td>201225</td><td></td></tr><tr><td>Forward Primer TPU1 (5&amp;acute;-AGA GTT TGA TCM TGG CTC AG-3&amp;rsquo;)</td><td>biomers.net, Ulm, Germany&amp;nbsp;</td><td>-</td><td></td></tr><tr><td>Reverse Primer RTU4 (5&amp;acute;-TAC CAG GGT ATC TAA TCC TGT T-3&amp;acute;)</td><td>biomers.net, Ulm, Germany&amp;nbsp;</td><td>-</td><td></td></tr><tr><td>Mastercycler&amp;nbsp;</td><td>Eppendorf, Hamburg, Germany</td><td>-&amp;nbsp;</td><td>Thermocylcer</td></tr><tr><td>Reaction tube 1.5 ml</td><td>SARSTEDT, N&amp;uuml;mbrecht, Germany</td><td>72,692</td><td></td></tr><tr><td>Reaction tube 2 ml</td><td>SARSTEDT, N&amp;uuml;mbrecht, Germany</td><td>72,693,005</td><td></td></tr><tr><td>PCR 8er-CapStrips</td><td>Biozym Scientific, Hessisch Oldendorf, Germany</td><td>711040X</td><td></td></tr><tr><td>PCR 8er-SoftStrips</td><td>Biozym Scientific, Hessisch Oldendorf, Germany</td><td>711030X</td><td></td></tr><tr><td>Sharp R-ZV11&amp;nbsp;</td><td>Sharp Electronics, Hamburg, Germany</td><td>-</td><td>Microwave</td></tr><tr><td>Titriplex III (EDTA Na&lt;sub&gt;2&lt;/sub&gt;-salt dehydrate; 1 kg)</td><td>Merck, Darmstadt, Germany</td><td>1084211000</td><td></td></tr><tr><td>SeaKem LE Agarose</td><td>Biozym Scientific, Hessisch Oldendorf, Germany</td><td>849006</td><td></td></tr><tr><td>SmartLadder SF - 100 to 1,000 bp</td><td>Eurogentec, L&amp;uuml;ttich, Belgium</td><td>MW-1800-04</td><td></td></tr><tr><td>Bromophenol blue (25 g)</td><td>Sigma-Aldrich Chemie, M&amp;uuml;nchen, Germany</td><td>B0126</td><td></td></tr><tr><td>Xylene cyanol FF (10 g)</td><td>Sigma-Aldrich Chemie, M&amp;uuml;nchen, Germany</td><td>X4126</td><td></td></tr><tr><td>ComPhor L Maxi&amp;nbsp;</td><td>Biozym, Hessisch Oldendorf, Germany</td><td>-</td><td></td></tr><tr><td>Ethidium bromide solution 1% (10 ml)</td><td>Carl Roth, Karlsruhe, Germany</td><td>2218.1</td><td></td></tr><tr><td>Gel Doc 2000</td><td>Bio-Rad Laboratories, M&amp;uuml;nchen, Germany</td><td>-</td><td>Gel-documentation system&amp;nbsp;</td></tr><tr><td>ExoSAP-IT (500 reactions)</td><td>Affymetrix UK, Wooburn Green, High Wycombe, United Kingdom</td><td>78201</td><td></td></tr><tr><td>Buffer (10x) with EDTA&amp;nbsp;</td><td>Life Technologies, Darmstadt, Germany</td><td>402824</td><td></td></tr><tr><td>BigDye Terminator Kit v1.1</td><td>Life Technologies, Darmstadt, Germany</td><td>4337450</td><td></td></tr><tr><td>Hi-Di formamide (25 ml)</td><td>Life Technologies, Darmstadt, Germany</td><td>4311320</td><td></td></tr><tr><td>DyeEx 2.0 Spin Kit (250)</td><td>Qiagen, Hilden, Germany</td><td>63206</td><td></td></tr><tr><td>3130 Genetic Analyzer</td><td>Life Technologies, Darmstadt, Germany</td><td>-</td><td>Sequencer</td></tr><tr><td>MicroAmp optical 96-well reaction plate with barcode</td><td>Life Technologies, Darmstadt, Germany</td><td>4306737</td><td></td></tr><tr><td>3130 Genetic Analyzer, plate base 96-well</td><td>Life Technologies, Darmstadt, Germany</td><td>4317237</td><td></td></tr><tr><td>3130 Genetic Analyzer, plate retainer 96-well</td><td>Life Technologies, Darmstadt, Germany</td><td>4317241</td><td></td></tr><tr><td>3130 Genetic Analyzer, well plate septa</td><td>Life Technologies, Darmstadt, Germany</td><td>4315933</td><td></td></tr><tr><td>3130 Genetic Analyzer, POP-7 Polymer, 7 ml</td><td>Life Technologies, Darmstadt, Germany</td><td>4352759</td><td></td></tr><tr><td>3130 Genetic Analyzer, 4-Capillary Array, 50 cm</td><td>Life Technologies, Darmstadt, Germany</td><td>4333466</td><td></td></tr><tr><td>Sequencing Analysis Software 5.4</td><td>Life Technologies, Darmstadt, Germany</td><td>-</td><td></td></tr><tr><td>microflex</td><td>Bruker Daltonik, Bremen, Germany</td><td>-</td><td>Our mMIS</td></tr><tr><td>VITEK MS</td><td>bioM&amp;eacute;rieux, N&amp;uuml;rtingen, Germany&amp;nbsp;</td><td></td><td>2&lt;sup&gt;nd&lt;/sup&gt; mMIS</td></tr><tr><td>flexControl 3.4</td><td>Bruker Daltonik, Bremen, Germany</td><td>-</td><td>Device control software</td></tr><tr><td>Biotyper Realtime Classification 3.1 (RTC)</td><td>Bruker Daltonik, Bremen, Germany</td><td>-</td><td>Analysis software</td></tr><tr><td>&amp;alpha;-cyano-4-hydroxycinnamic acid, HCCA, 1&amp;nbsp;g</td><td>Bruker Daltonik, Bremen, Germany</td><td>201344</td><td></td></tr><tr><td>Peptide Calibration Standard II</td><td>Bruker Daltonik, Bremen, Germany</td><td>222570</td><td></td></tr><tr><td>MSP 96 target polished steel</td><td>Bruker Daltonik, Bremen, Germany</td><td>8224989</td><td></td></tr><tr><td>peqGREEN</td><td>peqlab&amp;nbsp;</td><td>37-5010</td><td></td></tr><tr><td>MALDI Biotyper Galaxy&amp;nbsp;</td><td>Bruker Daltonik, Bremen, Germany</td><td>Part No. 1836007&amp;nbsp;</td><td>Automated sample preparation system</td></tr><tr><td>Vitek 2&amp;nbsp;</td><td>bioM&amp;eacute;rieux, N&amp;uuml;rtingen, Germany&amp;nbsp;</td><td></td><td>Our aMIS</td></tr><tr><td>MicroScan&amp;nbsp;</td><td>Beckman Coulter, Krefeld, Germany</td><td></td><td>2&lt;sup&gt;nd&lt;/sup&gt; aMIS</td></tr><tr><td>BD Phoenix&amp;trade; Automated Microbiology System</td><td>BD, Heidelberg, Germany</td><td></td><td>3&lt;sup&gt;rd&lt;/sup&gt; aMIS</td></tr><tr><td>&lt;i&gt;Staphylococcus aureus&lt;/i&gt;&amp;nbsp;subsp.&amp;nbsp;aureus&amp;nbsp;Rosenbach (ATCC&amp;reg;&amp;nbsp;25923&amp;trade;)</td><td>American Type Culture Collection (ATCC), LGC Standards, Molsheim, France</td><td></td><td>Positive control for PCR&amp;nbsp;</td></tr></tbody></table>

identification of rare bacterial pathogens by 16s rrna gene sequencing and maldi-tof ms

有许多的这些报告导致特别是在免疫功能低下患者严重感染罕见的，因此，不能充分描述的细菌病原体。在大多数情况下只有很少的数据，主要是出版病例报告，提供其研究这些病原体的作用，作为一种传染性病原体。因此，为了阐明这些微生物的致病性质，有必要进行的流行病学研究，其中包括大量的这些细菌。在这样的监控研究中所用的方法必须满足以下条件:菌株的识别必须根据有效命名法准确，它们应该是易于处理（鲁棒性），经济的常规诊断和他们要产生可比不同实验室之间的结果。通常，有一种用于在常规设置识别细菌菌株三种策略:1）的表型鉴定表征Biochemica公司细菌的升和代谢性质，2）分子技术如16S rRNA基因测序和3）质谱作为一种新的蛋白质为基础的方法。因为质谱和分子的方法可用于鉴定了大量的各种细菌物种的最有前途的工具，描述这两种方法。使用这些技术时进步，局限性和潜在的问题进行了讨论。

MALDI-TOF MS是一种新型的，用于微生物常规诊断快速和廉价的方法。通过MALDI-TOF MS的菌种鉴定产生光谱主要由核糖体蛋白，还包括其他"与受影响到环境条件的最小程度看家功能非常保守的蛋白"的这个MMIS的17 .The数据库包含一大组参考光谱的甚至目前在临床分离株很少发现细菌能够安全地识别7,56,57。得分值显示所识别的物种的可靠性。上述2.300分数代表，很?...

Watch this Scientific Journal Video about Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS at JoVE.com

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

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.

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

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

identification-rare-bacterial-pathogens-16s-rrna-gene-sequencing

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

17.7K 次观看.  TU Dresden. 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.

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

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics (MSPP)

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. Allelic isoforms of the detectable biomarker ions result in isolate-specific mass shifts. Mass spectrometry-based phyloproteomics (MSPP) is a novel technique that combines the mass spectrometric detectable biomarker masses in a scheme that allows deduction of phyloproteomic relations from isolate specific mass shifts compared to a genome sequenced reference strain. The deduced amino acid sequences are then used to calculate MSPP-based dendrograms.
Here we describe the workflow of MSPP by typing a Campylobacter jejuni ssp. doylei isolate collection of seven strains. All seven strains were of human origin and multilocus sequence typing (MLST) demonstrated their genetic diversity. MSPP-typing resulted in seven different MSPP sequence types, sufficiently reflecting their phylogenetic relations.
The C. jejuni ssp. doylei MSPP scheme includes 14 different biomarker ions, mostly ribosomal proteins in the mass range of 2 to 11 kDa. MSPP can in principle, be adapted to other mass spectrometric platforms with an extended mass range. Therefore, this technique has the potential to become a useful tool for strain level microbial typing.

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics MSPP

Mass spectrometry-based phyloproteomics (MSPP) was used to type a collection of Campylobacter jejuni ssp. doylei isolates at the strain level in comparison to multilocus sequence typing (MLST).

器的子类型<em&gt;空肠弯曲菌</em&gt; SSP。<em&gt; doylei</em&gt;分离株使用基于光谱质谱PhyloProteomics（MSPP）

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. ...

科研

遗传学

Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, we developed IDBac&#8212;a low-cost and high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bioinformatics pipeline. IDBac software is designed for non-experts, is freely available, and capable of analyzing a few to thousands of bacterial colonies. Here, we present procedures for the preparation of bacterial colonies for MALDI-TOF MS analysis, MS instrument operation, and data processing and visualization in IDBac. In particular, we instruct users how to cluster bacteria into dendrograms based on protein MS fingerprints and interactively create Metabolite Association Networks (MANs) from specialized metabolite data.

IDBac is an open-source mass spectrometry-based bioinformatics pipeline that integrates data from both intact protein and specialized metabolite spectra, collected on cell material scraped from bacterial colonies. The pipeline allows researchers to rapidly organize hundreds to thousands of bacterial colonies into putative taxonomic groups, and further differentiate them based on specialized metabolite production.

使用开源 MALDI TOF-MS IDBac 管道分析微生物蛋白和专用代谢物数据

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, ...

生物化学

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

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 &#215; 105-1.2 &#215; 109 CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of &#8805;0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae,&#160;and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (&#946;-lactams), aadA, aac (6&#39;)-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, &#946;-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the&#160;chloramphenicol acetyltransferase gene catB10, and&#160;the rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.

Here, we present a detailed protocol for the isolation and identification of antibiotic-resistant bacteria from water and the molecular characterization of their antibiotic resistance genes (ARGs). The use of culture-based and non-culture-based (metagenomic analysis) techniques provides complete information about the total bacterial diversity and the total pool of different ARGs present in freshwaters from Mumbai, India.

水性抗生素耐药细菌的分离和鉴定及其抗生素耐药基因的分子表征

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

免疫与感染

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain using antibiotic induction, and identified by MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. The immunity protein of colicin E3 (Im3) and the immunity protein of bacteriocin (Im-Bac) were identified from prominent b- and/or y-type fragment ions generated by the polypeptide backbone cleavage (PBC) on the C-terminal side of aspartic acid, glutamic acid, and asparagine residues by the aspartic acid effect fragmentation mechanism. The software rapidly scans in silico protein sequences derived from the whole genome sequencing of the bacterial strain. The software also iteratively removes amino acid residues of a protein sequence in the event that the mature protein sequence is truncated. A single protein sequence possessed mass and fragment ions consistent with those detected for each immunity protein. The candidate sequence was then manually inspected to confirm that all detected fragment ions could be assigned. The N-terminal methionine of Im3 was post-translationally removed, whereas Im-Bac had the complete sequence. In addition, we found that only two or three non-complementary fragment ions formed by PBC are necessary to identify the correct protein sequence. Finally, a promoter (SOS box) was identified upstream of the antibacterial and immunity genes in a plasmid genome of the bacterial strain.

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

Here we present a protocol for the rapid identification of proteins produced by genomically sequenced pathogenic bacteria using MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. Metastable protein ions fragment because of the aspartic acid effect and this specificity is exploited for protein identification.

使用MALDI-TOF-TOF-MS/MS和自上而下的蛋白质组学分析鉴定 大肠杆菌 中的抗菌免疫蛋白

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain ...

化学

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.

的16S核糖体RNA基因的扩增子的新一代测序

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 ...

生物学

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

使用MALDI-TOF质谱和自定义数据库的表征细菌土著一个独特的洞穴环境（卡切内岩洞，AZ，USA）

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

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.

16S rRNA-扩增子测序法对粪便微生物特性的指导协议

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

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.

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

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

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

摘要

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此视频中的章节

器的子类型

使用开源 MALDI TOF-MS IDBac 管道分析微生物蛋白和专用代谢物数据

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

水性抗生素耐药细菌的分离和鉴定及其抗生素耐药基因的分子表征

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

使用MALDI-TOF-TOF-MS/MS和自上而下的蛋白质组学分析鉴定大肠杆菌中的抗菌免疫蛋白

的16S核糖体RNA基因的扩增子的新一代测序

使用MALDI-TOF质谱和自定义数据库的表征细菌土著一个独特的洞穴环境（卡切内岩洞，AZ，USA）

16S rRNA-扩增子测序法对粪便微生物特性的指导协议

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

摘要

探索更多视频

此视频中的章节

器的子类型

使用开源 MALDI TOF-MS IDBac 管道分析微生物蛋白和专用代谢物数据

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

水性抗生素耐药细菌的分离和鉴定及其抗生素耐药基因的分子表征

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

使用MALDI-TOF-TOF-MS/MS和自上而下的蛋白质组学分析鉴定 大肠杆菌 中的抗菌免疫蛋白

的16S核糖体RNA基因的扩增子的新一代测序

使用MALDI-TOF质谱和自定义数据库的表征细菌土著一个独特的洞穴环境（卡切内岩洞，AZ，USA）

16S rRNA-扩增子测序法对粪便微生物特性的指导协议

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

使用MALDI-TOF-TOF-MS/MS和自上而下的蛋白质组学分析鉴定大肠杆菌中的抗菌免疫蛋白