Name: genotyping of staphylococcus aureus by ribosomal spacer pcr (rs-pcr)
Uploaded: 2016-11-04T13:00:00.000+00:00
Duration: 8 min 51 s
Description: Watch this Scientific Journal Video about Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR at JoVE.com

The author thanks R. Boss, A. Cosandey, C. Fournier, I. Ivanovic, and J. Naskova for their excellent work.

1. 金黄色葡萄球菌菌株<ol><li>价差10微升的S.金黄色葡萄球菌的库存培养或一个菌落生长在含5％绵羊血（BA）哥伦比亚琼脂板上的选择性培养基上。 </li><li>在37℃需氧温育18小时至24小时。 </li></ol> 2. DNA提取<ol><li>制备含10mM的Tris-HCl和10mM 的 Na 2 EDTA，pH 8.5的TEL缓冲液中。在121℃制备等分试样并高压灭菌15分钟。 </li><li>用100微升TEL无菌塑料环或无菌的木制牙签，重悬收集BA 1殖民地。应用1.5 mL管与螺旋盖。 </li><li>孵育在95℃下进行10分钟。随后，立即将样品在湿冰。 </li><li>稀释样品1:100在适合的PCR（=模板DNA用于PCR） 的 H 2 O操作。非稀释和稀释的样品可以保持在-20℃下1个月。 </li></ol> 3. RS-PCR <ol><lI&gt;对于每个样品吸管成PCR管1.5微升的 H 2 0，12.5微升的Taq主混合物，2微升G1引物10μM的，2微升的L1引物10微米，7微升，对应于约30毫微克纯的模板DNA DNA 2。 </li><li>在标准PCR循环仪运行下面循环的程序:15分钟95℃，随后27个循环，包括94℃1分钟，随后在2分钟坡道和退火在55℃7分钟。再过2分钟斜坡后，在72℃延伸2分钟。通过在72℃最后延伸10分钟，然后冷却至4℃终止的PCR。 注:PCR产物可被储存在-20℃下5天。 </li><li>对于每次运行的RS-PCR包括一个阳性对照， 即，与已知的基因型（通常GTB）的样品，和一个阴性对照（H 2 O）。 </li></ol> 4.电泳<ol><li>使用脱氧核糖核酸（救护包）商用微型电泳试剂盒一起连接到PC和安装软件生物分析仪。仔细研究制造商的相应手册。 </li><li>设置在芯片涂刷站并根据制造商的手册调节注射器剪辑。 </li><li>根据制造商的手册制备凝胶染料混合。 </li><li>载入凝胶染料混合物（根据制造商的手册）。 <ol><li>平衡凝胶染料混合至RT使用前30分钟。 </li><li>提出了新的DNA芯片的芯片吸站。吸管9微升凝胶染料在井标记G. </li><li>确保柱塞被定位以1ml，然后关闭该芯片涂刷站。按柱塞，直到它被注射器夹子保持。正是等待30秒再松开注射器夹。 </li><li>等待5秒。活塞慢慢回拉至1ml的位置。 </li><li>打开芯片吸站和吸管9微升凝胶染料在井都标有&#39;G&#39;。 </li></ol></li><li>加载并购arker <ol><li>吸取5微升标记在所有12个样品孔，并在梯井。不要让这些13口井空的。 </li></ol></li><li>加载梯和样品。 <ol><li>吸管1μl的DNA梯状到良好标有阶梯符号。 </li><li>吸取1微升样品（用井）或1微升去离子水（未使用的水井）的。 </li><li>横过来时，芯片的适配器和涡旋1分钟2400转。 </li><li> 5分钟内运行在生物分析仪芯片。 </li></ol></li><li>运行分析<ol><li>启动生物分析仪和电脑相连。打开软件。 </li><li>打开生物分析仪的盖和芯片放入芯片保持器（芯片只适合单程）。仔细盖上盖子。 </li><li>在软件的仪器上下文菜单中选择"电泳"&gt;"双链"&gt;"DNA 7500"。接受软件的当前文件前缀或修改。</li><li>点击存在于软件的启动按钮。在软件中输入样本名。当芯片运行结束（后约30分钟） 运行邮件的末尾出现在生物分析仪软件。 </li><li>根据制造商的指示删除该芯片和清洁生物分析仪的电极。 </li></ol></li></ol> 5.从电泳图谱推断基因型<ol><li>加载泰姬陵软件（软件是免费提供从作者）。 </li><li>进口参考数据到泰姬陵软件:"文件"&gt;"导入参考数据" </li><li>打开在生物分析仪软件的数据上下文中运行电泳。选择一个样品的电泳轮廓。 </li><li>检查相关的峰值。 <ol><li>使用泰姬陵软件来检查相关峰:"工具"&gt;"平均荧光"。插入"荧光"框荧光值表示为荧光单位（FU），因为它们可以从生物分析仪软件被读出。将4（3）峰升序排列最高的荧光，以逗号分隔。例如:126130132137。 注意:如果不是所有的这些峰FU的由生物分析仪软件的指示，就需要通过从积读出来进行估计。 </li></ol></li><li>按"计算"按钮。如果观察到的荧光超过盒子"最小峰高"给出的值峰值是相关的。 </li><li>推断基因型。 <ol><li>插入文本框泰姬陵"输入峰（BP）"，在升序排列的所有相关峰基点大小和以逗号分隔。例如:440,462,519,579,637。 注意:如果不是所有这些峰的大小是由生物分析仪软件的指示，就需要通过从积读出来进行估计。 </li></ol></li><li>按"计算"按钮。 </li><li>在列表中，查找最小平方马氏距离"D2M"。如果相应的P值给它是≥0.05则概率为≥5％，该零假设H 0被拒绝（H 0:所观察到的与参考轮廓是相同的），这意味着所观察到的信息被接受，以完全相同指定基因型的基准轮廓。 </li><li>如果D2M是最小的，但相应的P值的字段是空的，拒绝H 0的概率为&lt;5％。 注意:这意味着该轮廓是不相等的，尽管D2M是最小的。这样的结果，可能会导致从非平均的电泳条件。 <ol><li>为了纠正这些偏差，插入"观察峰（BP）"值395的文本框，然后按"计算"按钮。再次检查最小D2M和相应的P值≥0.05。如果不成功，尝试值405，415，和425。 注意:有时更小的步骤，甚至是表示。如果仍然没有成功，所观察到的信息中不存在的参考数据，它可能是一个新的基因型或变体。 </li></ol></li><li>各种型材<ol><li>重复步骤对每个电泳图谱5.6至5.9分开。 </li></ol></li></ol>

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The authors have nothing to disclose.

整个过程中最关键的步骤是当在RS-PCR（&gt; 30纳克纯的DNA /测定）过量的DNA的2。在一般情况下，一个轮廓的分辨率为最佳，如果所有其峰在基线开始和结束。如果两个峰值靠得太近，分辨率是不完整的。在这些条件下，从而需要人工识别的生物分析仪软件可能导致不适当的峰鉴定。 所有表现为BP或FU的明显分离，相关峰作进一步的分析。太多模板DNA RS-PCR结果在宽峰，因此峰值峰重叠和受损的分辨率。此外，迁移较慢领先峰值大小被虚假增加，使得基因型不再可以推断。最后，不相关峰的数量较大。对于具有3个以上的山峰分布，最大荧光峰值通常不应超过150 FU。由RS-PCR基因分型如所述由事实，即在机投资是必要的，并在需要自由陵软件限定。事实上，一个标准的PCR仪和生物分析仪是必要的。前者是相当便宜的现今，而后者是更昂贵的。基于琼脂糖电泳的分辨率是不够的，即使使用高分辨率琼脂糖。在这些条件下，这只是可能GTB，GTC和所有其它基因型之间进行区分。使用MED试剂盒或琼脂糖电泳电泳的费用，但是，是相似的。与生物分析仪进一步的救护包一起优于在某种程度上标准琼脂糖凝胶电泳，后者的方法是很方便，直线前进，并取得数据中存在于简化了存储和进一步分析大大电子形式。主要所有市售bioanalyzers适于这种类型的分析提供的electrophoretIC分辨率是足够的，得到的条带的大小是准确和客观公正。 RS-PCR对S的牛株分辨率金黄色葡萄球菌是很高的。它是作为水疗打字一样好，而比MLST和电泳4,14更好。此外，相对于所有常用的亚型S的其他分型方法金黄色葡萄球菌 （ 温泉打字，MLST，PFGE），其样品通量高:DNA提取是方便，快捷，96个样品可以在一个RCR运行（通常是O / N）和电泳处理和基因型推理是直线前进。 96个样品的完整分析需要PCR一晚有一天电泳和评估。通过Fournier 等人的 2个 RS-PCR的其他优点是成本低，特别是当相比MLST需要7个基因在两个方向15进行测序。此外，RS-PCR是菌株的唯一方法预测contagiosity和致病参与牛金黄色葡萄球菌乳腺炎2,5，临床兽医学关键预测指标。最后，RS-PCR本身就足以调查的牛链球菌流行病学金黄色葡萄球菌 3,4,5,14。对数据的解释是直线前进，甚至在国际范围内，因为只有少数主要基因型观察2,3。对牛和人的菌株，RS-PCR和温泉打字之间流行病学比较一起优选作为后一种方法被广泛地用于亚型的人毒株，使得大量的数据是可用的。 MLST，但是，是适于评估菌株15的克隆能力和系统发育分析4。 有关的PCR仪故障排除，救护包，以及生物分析仪，相应的手册都将进行协商。在RS-PCR法的情况下，故障处理大量简化，如果适当的阳性和阴性的PCR对照是增量luded每次运行。如果阳性对照是否定的，PCR混合物没有充分组成和/或对照DNA被降解。如果阴性对照（H 2 O）产生的一个或多个带，所述PCR混合物中混有一些的DNA。在这两种情况下，配置文件不得解释和RS-PCR具有重复。如果解释是因为电泳问题是不可能的，电泳需要使用新的芯片进行重复。电泳问题通常特征在于迁移和一个对准不充分或不适当的装载和芯片的操纵导致两个标记带。如果获得的电泳图谱不能由陵软件来解释，由生物分析仪软件产生的资料文件被发送给作者以进行进一步评估。通常情况下，使用过多的模板DNA RS-PCR或情景代表了一种新的基因型或基因型变体。

葡萄球菌 （ 金黄色葡萄球菌 ）被称为全球用于在牛1乳房内感染（IMI）负责的最重要的病原体。在12个欧洲国家的奶牛研究由Fournier 等人 2瑞士奶牛示范和研究通过Cosandey 等 3和老板等 4 S.金黄色葡萄球菌从牛隔离IMI是一种遗传异质组。由16S-23S rRNA基因间区（RS-PCR）的PCR扩增，共有17个基因型在101流行病学独立的分离2最初检测。基因型B和C型（GTC）是最常见的（80％），而其它15个基因型（GTS）发生很少，各使所有的菌株1至4％。在欧洲3级，GTB，GTC，GTF，GTI和GTR是由所有分析的株（N = 456）的76％，最重要的基因型。 GTB是位于欧洲中部W¯¯hereas其他基因型广泛传播。菌株的其余24％包含41种基因型，其中许多，但是，观察到只有一次。 通过Fournier 等人的 2，Cosandey 等 3和格雷伯等人 5的研究进一步表明，基因型与它们的毒力基因图案在瑞士高度相关的，以及如在欧洲一级。该研究进一步揭示之间S.在IMI患病率差异巨大的金黄色葡萄球菌 GTB，GTC和其他基因型2,5:考虑GTB，最多在牛群奶牛的87％是由这种基因型感染。在GTC和其他基因型的情况下，然而，IMI仅在1至2的奶牛畜群的发现。 IMI引起S.黄色葡萄球菌 ，通常会导致在相应的乳腺炎症和牛奶中的增加的炎性细胞。因此，体细胞共牛奶UNTS（SCC），牛奶的品质在大多数国家的关键指标，增加从而导致降低牛奶价格甚至停止发货。 除了Fournier 等人 2的RS-PCR其他分型方法已为亚型S的牛菌株描述金黄色葡萄球菌 8-11。两种最常见的包括水疗打字12多位点序列分型（MLST）13。前者是基于DNA测序葡萄球菌温泉基因编码蛋白质A的可变间隔区，而后者要求7管家基因的测序。这意味着一个12和七个PCR的13，分别需要执行，随后扩增子纯化，测序反应和分析使用特定的设备。相比的RS-PCR，这些方法需要在导致低的样品通量和成本高的实验室一个相当大的额外的努力。该吞吐量是特别低PFGE。考虑到这些方法对S的牛株分辨率金黄色葡萄球菌 ，RS-PCR是至少作为温泉打字4和优于MLST 4或电泳15一样好。 所有这些方法包括二进制打字13表现在获得进一步的深入了解造成S.牛IMI的发病机制及其有效性所提到的限制的金黄色葡萄球菌 ，但因为它们是用于大型临床研究较少为宜。另外，作为由Fournier 等人描述由RS-PCR基因分型。2允许流行病学和S的致病潜力的可靠的预测金黄色葡萄球菌参与牛IMI，在临床兽医学两个关键值。

<table><tbody><tr><td>Tris(hydroxymethyl)-aminomethan</td><td>Merck</td><td>1.08382.0500</td><td>Mw = 121.14 g/mol</td></tr><tr><td>Titriplex III</td><td>Merck</td><td>1.08418.0250</td><td>Mw = 372.24 g/mol; Substance is equivalent to Na&lt;sub&gt;2&lt;/sub&gt;EDTA&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Hydrochloric acid 5 mol/L</td><td>Merck</td><td>1.09911.0001</td><td></td></tr><tr><td>Columbia agar+5% sheep blood</td><td>bioM&amp;eacute;rieux</td><td>43049</td><td></td></tr><tr><td>Agilent DNA7500 Kit</td><td>Agilent Technologies</td><td>5067-1504</td><td></td></tr><tr><td>Agilent 2100 Bioanalyzer</td><td>Agilent Technologies</td><td>G2940CA</td><td></td></tr><tr><td>Agilent 2100 expert sofware</td><td>Agilent Technologies</td><td></td><td></td></tr><tr><td>HotStarTaq master mix&amp;nbsp;</td><td>Qiagen</td><td>203445</td><td></td></tr><tr><td>Primer L1</td><td>Mycrosinth</td><td></td><td>5&#039;CAA GGC ATC CAC CGT3&#039;</td></tr><tr><td>Primer G1</td><td>Mycrosinth</td><td></td><td>5&#039;GAA GTC GTA ACA AGG3&#039;</td></tr></tbody></table>

genotyping of staphylococcus aureus by ribosomal spacer pcr (rs-pcr)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

基因型及其变体的定义电泳轮廓从所有确定的基因型在一个以上的频带不同的被认为是新的基因型。新基因型根据Fournier 等人的命名和扩展。5通往基因型GTA到GTZ，其次是基因型GTAA到GTAZ，并GTBA到GTBZ和GTCA到GTCC目前。只在一个频带变化被视为一个基因型变体。它的标志是基因型（如 GTRI，GTRII）的名称后，上标罗马数字。总共，目前有141基因型和变体在基准数据的基础上。 根据以上所述，RS-PCR由Fournier 等人 5的条件是高度重现基因型链球菌菌株金黄色葡萄球菌 。一个明确的鉴定已知基因型或变体始终是可能的，如果技术问题可被排除。典型的结果示于图1中展示的12的RS-PCR产物的电泳和在由生物分析仪软件伪凝胶模式呈现。每个泳道的特征在于在最后两个或更多个PCR条带的图案，并在前面的标记带和标记带。双击进入车道将打开一个单独的窗口对应的，实际测得的电泳轮廓。生物分析仪软件允许除其他的可能性，正在读取的观察到的峰的大小为荧光单位FU（ 图2）或在碱基（ 图3），由此一个峰在伪凝胶模式精确地对应于一个波段。在傅峰值读数需要评估在陵软件简档的相关峰（ 图4），在碱基阅读推断基因型。无关峰在泰姬陵软件小豌豆定义KS的观测荧光FU低于具有最高荧光的4（3）的峰的几何平均值的40％。当在RS-PCR过量的DNA（&gt; 30纳克纯的DNA /测定）它们通常观察到2。 作为一个例子，存在于图1中样品211的基因型推断。 图2中的电泳轮廓（生物分析仪软件）和分析使用该工具来测试无关峰泰姬陵软件的可视化分析发现6 395基点，432基点，453基点，505基点，567基点的峰值大小相关的峰值，和622碱基（ 图3）。这些值被填充并在文本"输入峰（bp）的"的陵软件（ 图4）由逗号分隔。为了校正特定的运行的偏差，该值415被插入到"看峰（bp）的"（ 图4）的文本框。具有按"℃后alculate"按钮，则提出的名单由基因型与查询和指示基因型轮廓之间的平方马哈拉诺比斯距离D2M一起排序。滚动列表寻找最小D2M。在目前情况下，最小的D2M等于4.499（ 图4）与P≥0.05的值，这意味着查询轮廓不显著从GTB之一不同。 基因型变体表示为_I在陵软件_XV。在B型的情况下，存在三种变体GBT 我 ，GBT II和GBTⅢ示为B_I，B_II和B_III在软件（ 图4）。 救护包的芯片允许12 RS-PCR分析的产品的时候。如果较少的产品都可用，剩余孔需要被装入去离子水。结果可能只有控制来解释我在RS-PCR ncluded是适当的。 <img alt="图1" src="/files/ftp_upload/54623/54623fig1.jpg" /> S. 图1.代表12 RS-PCR产品及其推断基因型伪凝胶。十二个不同的菌株金黄色葡萄球菌是由RS-PCR一起使用MED套件与生物分析仪及附带的软件进行分析。在左侧的车道显示了系统在碱基对大小校准标记或"阶梯"。在图形中，样品名称和相应的基因型起诉的泰姬陵软件获得的顶端。 <a href="https://www.jove.com/files/ftp_upload/54623/54623fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54623/54623fig2.jpg" /> 图2. SA电泳简介mple 161存在于图1.在测量荧光在FU表示的峰的顶部。这不是由生物分析仪软件需要检测出视觉上区分峰由作为与113 FU（蓝色）的估计荧光值高峰期进行的阴谋读出来进行估算。 <a href="https://www.jove.com/files/ftp_upload/54623/54623fig2large.jpg" target="_blank">请点击此处查看这个大版本数字。</a> <img alt="图3" src="/files/ftp_upload/54623/54623fig3.jpg" /> 图3. 样品211出现在图1中它们的大小在BP表示峰顶部的电泳轮廓 。 <a href="https://www.jove.com/files/ftp_upload/54623/54623fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" sRC ="/文件/ ftp_upload / 54623 / 54623fig4.jpg"/&gt; 图4. 屏幕截图泰姬陵软件。在文本框中输入"输入峰（BP）"从图3的样品211的峰值大小填写。 <a href="https://www.jove.com/files/ftp_upload/54623/54623fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR at JoVE.com

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

基因分型<em&gt;金黄色葡萄球菌</em&gt;由核糖体垫片的PCR（RS-PCR）

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

genotyping-of-staphylococcus-aureus-by-ribosomal-spacer-pcr-rs-pcr

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

9.6K 次观看.  Agroscope. Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

视频: Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

无基因克隆的基因破坏的变形链球菌菌株的生成

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

科研

遗传学

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

为期一天的血培养的细菌病原体检测和耐药性检测的工作流程计划

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

免疫与感染

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

生物传感器检测抗生素耐药金黄色葡萄球菌细菌

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

生物工程

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

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Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5&#8217; extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.

MAPS technology has been developed to scrutinize the targetome of a specific regulatory RNA in vivo. The sRNA of interest is tagged with a MS2 aptamer enabling the co-purification of its RNA partners and their identification by RNA sequencing. This modified protocol is particularly suited for Gram-positive bacteria.&#160;

MS2亲和力纯化与革兰阳性细菌中的RNA测序相结合

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized ...

生物化学

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

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

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

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 ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

全基因组基因缺失<em&gt;链球菌血统</em通过高通量PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

生物学

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 ...

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

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

高效的核酸提取和16S rRNA基因序列的细菌群落特征

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

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 ...

基因分型

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