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。

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			<td>CHROMASOLV, HPLC grade water, 1 L</td>
			<td>Sigma-Aldrich Chemie, M&#252;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 1,0mm</td>
			<td>VWR International, Darmstadt, Germany</td>
			<td>412-2917</td>
			<td></td>
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		<tr>
			<td>Thermomixer 5436</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>2050-100-05</td>
			<td></td>
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		<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 (1000 U)</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>201225</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Forward Primer TPU1 (5&#180;-AGA GTT TGA TCM TGG CTC AG-3&#8217;)</td>
			<td rowspan="2">biomers.net GmbH, Ulm, Germany&#160;</td>
			<td rowspan="2">-</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Reverse Primer RTU4 (5&#180;-TAC CAG GGT ATC TAA TCC TGT T-3&#180;)</td>
			<td rowspan="2">biomers.net GmbH, Ulm, Germany&#160;</td>
			<td rowspan="2">-</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
		</tr>
		<tr>
			<td>Mastercycler&#160;</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>-&#160;</td>
			<td>Thermocylcer</td>
		</tr>
		<tr>
			<td>Reaction tube 1.5 mL</td>
			<td>SARSTEDT, N&#252;mbrecht, Germany</td>
			<td>72,692</td>
			<td></td>
		</tr>
		<tr>
			<td>Reaction tube 2 mL</td>
			<td>SARSTEDT, N&#252;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&#160;</td>
			<td>Sharp Electronics, Hamburg, Germany</td>
			<td>-</td>
			<td>Microwave</td>
		</tr>
		<tr>
			<td>Titriplex III (EDTA Na2-salt dehydrate; 1 kg)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1084211000</td>
			<td></td>
		</tr>
		<tr>
			<td>SeaKem LE Agarose</td>
			<td rowspan="2">Biozym Scientific, Hessisch Oldendorf, Germany</td>
			<td rowspan="2">849006</td>
			<td></td>
		</tr>
		<tr>
			<td>(2 x 500 g)</td>
			<td></td>
		</tr>
		<tr>
			<td>SmartLadder SF - 100 to 1000 bp</td>
			<td>Eurogentec, L&#252;ttich, Belgium</td>
			<td>MW-1800-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromphenol blue (25 g)</td>
			<td>Sigma-Aldrich Chemie, M&#252;nchen, Germany</td>
			<td>B0126</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene cyanol FF (10 g)</td>
			<td>Sigma-Aldrich Chemie, M&#252;nchen, Germany</td>
			<td>X4126</td>
			<td></td>
		</tr>
		<tr>
			<td>ComPhor L Maxi&#160;</td>
			<td>Biozym, Hessisch Oldendorf, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium bromide solution 1&#160;%(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&#252;nchen, Germany</td>
			<td>-</td>
			<td>Gel-documentation system&#160;</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 (10 x) with EDTA&#160;</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>Sequenzer</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 (the MALDI TOF MS maschine)</td>
			<td>Bruker Daltonik, Bremen, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI Biotyper (the MALDI TOF MS system)</td>
			<td>Bruker Daltonik, Bremen, Germany</td>
			<td>-</td>
			<td>our mMIS</td>
		</tr>
		<tr>
			<td>VITEK MS&#160;</td>
			<td>bioM&#233;rieux, N&#252;rtingen, Germany&#160;</td>
			<td></td>
			<td>2nd mMis&#160;</td>
		</tr>
		<tr>
			<td>flexControl 3.4 (control software)</td>
			<td>Bruker Daltonik, Bremen, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotyper Realtime Classification 3.1 (RTC), (analysis software)</td>
			<td>Bruker Daltonik, Bremen, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-cyano-4-hydroxycinnamic acid, HCCA, 1&#160;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&#160;</td>
			<td>peqlab&#160;</td>
			<td>37-5010</td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI Biotyper Galaxy&#160;</td>
			<td>Bruker Daltonik, Bremen, Germany</td>
			<td>Part No. 1836007&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitek 2&#160;</td>
			<td>bioM&#233;rieux, N&#252;rtingen, Germany&#160;</td>
			<td></td>
			<td>our aMis&#160;</td>
		</tr>
		<tr>
			<td>MicroScan&#160;</td>
			<td>Beckman Coulter&#160;</td>
			<td></td>
			<td>2nd aMis&#160;</td>
		</tr>
		<tr>
			<td>BD Phoenix&#8482; Automated Microbiology System</td>
			<td>BD</td>
			<td></td>
			<td>3rd aMis&#160;</td>
		</tr>
		<tr>
			<td>Staphylococcus aureus&#160;subsp.&#160;aureus&#160;Rosenbach (ATCC&#174;&#160;25923&#8482;)</td>
			<td>ATCC&#160;</td>
			<td></td>
			<td>postive control for PCR&#160;</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

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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

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

"发光法"的分类检测细菌病原体的记者噬菌体

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

科研

免疫与感染

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

斑马鱼的胚胎细胞内细菌病原体感染

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

单细胞测量液泡破裂引起细胞内病原体

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

全细胞MALDI-TOF质谱法是一种准确，快速的方法来分析巨噬细胞活化的不同模式

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and ...

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 ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

荧光标记基因的基因枪转化成机会性真菌病原<em&gt;新型隐球菌</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

原位检测使用地高辛标记的DNA探针石蜡包埋组织中的细菌靶向16S rRNA基因

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

在动物标本呼吸道病原体的高通量检测通过纳米PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

A Method for Targeted 16S Sequencing of Human Milk Samples

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as with all these technologies, reproducible results depend on a laboratory workflow that incorporates appropriate precautions and controls. This is particularly important with low-biomass samples where contaminating bacterial DNA can generate misleading results. This article details a semi-automated workflow to identify microbes from human breast milk samples using targeted sequencing of the 16S ribosomal RNA (rRNA) V4 region on a low- to mid-throughput scale. The protocol describes sample preparation from whole milk including: sample lysis, nucleic acid extraction, amplification of the V4 region of the 16S rRNA gene, and library preparation with quality control measures. Importantly, the protocol and discussion consider issues that are salient to the preparation and analysis of low-biomass samples including appropriate positive and negative controls, PCR inhibitor removal, sample contamination by environmental, reagent, or experimental sources, and experimental best practices designed to ensure reproducibility. While the protocol as described is specific to human milk samples, it is adaptable to numerous low- and high-biomass sample types, including samples collected on swabs, frozen neat, or stabilized in a preservation buffer.

A semi-automated workflow is presented for targeted sequencing of 16S rRNA from human milk and other low-biomass sample types.

人乳样品靶向16S 测序方法的研究

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