这项工作是支持的的AZM Profileringsfonds（PF245）。

第一部分：病原鉴定 1。样品制备 注：在以下协议所述的整个分子的工作流程应根据建议在分子诊断质量保证3。 <ol><li>加入血培养0.1毫升分装到0.9毫升0.9％至1.5 ml反应管的NaCl成为1:10的稀释后的样品。（稀为了防止定量PCR的抑制）。 </li><li> 13400 XG离心5分钟沉淀细菌的DNA样本。 </li><li>细菌沉淀重悬于100μL无菌矿物质的H 2 O </li><li>保存于4°C，直到进一步利用DNA样本。 </li></ol> 2。鉴定检测：实时的16S rDNA PCR技术<ol><li>准备如下的反应混合物。该法由四个不同的反应，每个样品。每个混合物包括12.50μL主混合，0.9微米正向引物（5-TCCTACGGGAGGCAGCAGT-3），7，0.6微米的反向引物（5-GGACTACCAGGGTATCTAATCCTGTT-3），8和面板的探头。探头的数量是每个在以下四个不同的反应。 </li><li>第一反应包括： <ul><li> 0.2μM的通用探头（5 FAM CGTATTACCGCGGCTGCTGGCAC-3-BHQ1）8 </li><li> 0.2μM的体育的铜绿探头（5 -乔- CCAAAACTACTGAGCTAGAGTACG-3-BHQ1） </li></ul></li><li>第二个反应包括： <ul><li> 0.2μM的大肠杆菌大肠杆菌探头（5 -乔- GGAGTAAAGTTAATACCTTTGCTCATT-3-BHQ1） </li><li> 0.2μM的假单胞菌 。探头（5-NED的CCTTCCTCCCAACTTAAAGTGCTT-3-MGBNFQ） </li></ul></li><li>第三个反应包括： <ul><li> 0.2μM的葡萄球菌 。探头（5-NED的AATCTTCCGCAATGGGCGAAAGC-3-MGBNFQ） </li><li> 0.2微米的S。金黄色葡萄球菌探头（5-FAM-AGATGTGCACAGTTACTTACACATAT-3-BHQ1） </li><li> 0.2μM的肠球菌 。 （5 - 乔 - TCCTTGTTCTTCTCTAACAACAGAG-3-BHQ1） </li></ul></li><li>第四反应包括： <ul><li> 0.2μM的通用探头（5-FAM-CGTATTACCGCGGCTGCTGGCAC-3-BHQ1） </li><li> 0.3μM的链球菌属。探头（5-NED的CCAGAAAGGGACSGCTAACT-3-MGBNFQ） </li><li> 0.2微米的S。肺炎探头（5 -乔- CCAAAGCCTACTATGGTTAAGCCA-3-BHQ1） </li></ul></li><li>加入无菌去离子H 2 O，达到总量的20μL。每个反应混合物中加入20微升至96孔PCR板的井。 </li><li>每孔加入5μL样品。 </li><li>使用胶膜​​密封的96孔PCR板。 </li><li>在ABI PRISM 7900HT实时定量PCR系统使用以下最佳的热循环条件下运行的板块： <ul><li>在50°C加热10分钟前</li><li>最初在95°C变性15分钟</li><li> 42个循环<ul type="circle"><li>在95°C变性15秒</li><li>在60°C退火1 min </li></ul></li></ul></li></ol> 3。结果分析<ol><li>在标签分析设置，调整阈值的CT分析0.1。缩小基线配置启动（循环）：6和尾（周期）：15。 </li><li>记录所有样品的循环阈值（CT）值。考虑PCR检测结果为阳性截止值可以设置为CT值35。血培养中存在的细菌数量为10 7 10 11 CFU /毫升，CT-值低于35的产生。 </li></ol>第二部分：药敏试验 4。血培养阳性9细菌的分离<ol><li>从阳性血培养瓶吸5毫升肉汤和转移到血清分离管。 </li><li> 2000年为10分钟XG离心血清分离管。 </li><li>倒掉上清血清分离管。 </li><li>转让BACteria用无菌棉签管凝胶层到0.9％的盐水，直到得到0.5麦克法兰标准悬挂。 </li></ol> 5。接种微滴度板<ol><li> 0.5麦克法兰悬挂在双集中穆勒韩丁第二肉汤稀释，以形成一个5×10 5 CFU / ml的悬浮。 </li><li>将此悬浮微滴度板含有抗生素的选择（ 见表1）井。 </li><li>在37°C孵育6小时微滴度板。 </li><li>储存在4°C（负增长控制）暂停等分。 </li><li>培养6小时后，转移到无菌管每口井的内容，以及负增长，控制样本被储存在4°C </li><li> 16000 XG 5分钟的离心管。 </li><li>小心取出上清液，不干扰细菌沉淀。 </li><li>在无菌矿物质的Ĥ悬浮颗粒<sUB&gt; 2 O。 </li><li> 10倍稀释样品在无菌矿物质的H 2 O </li></ol> 6。实时的16S rDNA PCR扩增10 <ol><li> PCR混合物准备如下： <ul><li> 12.50μL的iQ SYBR Green超</li><li> 0.5μM的正向引物16S-1（5-TGGAGAGTTTGATCCTGGCTCAG-3）11 </li><li> 0.25微米的反向引物16S-2（5 TACCGCGGCTGCTGGCAC-3）11 </li><li>无菌去离子H 2 O的总体积20μL </li></ul></li><li>加入20μlPCR混合物的96孔PCR板的井。 </li><li>每孔加入5μL样品。 </li><li>使用胶膜​​密封的96孔PCR板。 </li><li>运行盘上​​的MyiQ™实时单色实时PCR检测系统，使用以下最佳的热循环条件： <ul><li>最初在95°C变性4分钟</li><li>初始退火65℃30秒</li><li> 35个循环<ul><li>变性95℃15秒</li><li>在60°C退火1 min </li></ul></li><li>熔解曲线分析（60-95°C的20分钟增量为0.57°C） </li></ul></li></ol> 7。结果分析<ol><li>计算截止的Ct值，使用下列公式（抗生素类型而定）之一。 <ul><li>一般情况下： 截止Ct值= Ct值正增长，控制+ 0.5×（Ct值负增长控制- Ct值正增长，控制） </li><li>哌拉西林，哌拉西林/他唑巴坦和头孢他啶在革兰氏阴性菌棒，阿莫西林，苯唑西林的金黄色葡萄球菌的甲氧苄啶/磺胺甲恶唑;在肠球菌阿莫西林： 切断Ct值= Ct值正增长，控制+ 0.25×（Ct值出现负增长。控制- Ct值正增长，控制） </li></ul></li><li>使用培养用无菌去离子H 2 O为正增长，控制样品。</li><li>根据微生物，使用适当的负增长控制： <ul><li>革兰氏阴性菌棒样品的培养与抗生素的混合物</li><li> 肠球菌样品储存在4°C </li><li> 金黄色葡萄球菌样品储存在4°C </li></ul></li><li>确定的易感性（S）或电阻（R）的测试如下抗生素的应变： <ul><li>一个CT值高于截止Ct值表示易感性</li><li>一个CT值低于截止Ct值表示电阻</li></ul></li></ol> 8。代表结果两个模式生物，即革兰氏阴性菌E.大肠杆菌和革兰氏阳性与金黄色葡萄球菌 ，选择可视化合并过程的检测和识别细菌病原体和测定其抗菌简介。该协议的第一部分包括病原鉴定。 SPEcific探针被设计为8个临床相关的微生物的检测。在存在细菌面板中包含的目标，扩增曲线的生成和计算CT值（ 图2）。考虑PCR检测结果为阳性截止值设置为35 Ct值。 图2A中， 大肠杆菌感染的血培养鉴定的个人资料显示。 16S通用探针包含两个独立的反应混合物，从而产生两个扩增曲线（25.20和25.95克拉）。第三个信号是来自E.探头具体大肠杆菌 （CT，27.04）。一个与鉴定金黄色葡萄球菌感染的血培养如图2B所示。 16S通用探针33.35和33.71的放大信号。剩下的两个信号是来自特定的探针葡萄球菌 。 和 S 金黄色葡萄球菌 （32.48和30.59克拉）。 图3是一种抗生素药敏试验扩增曲线代表大肠杆菌也显示，例如图2A中。每一行代表一个抗生素的细菌样本培养。低CT值的样本是在增长中存在一种抗生素，表明电阻测试抗生素发生的样本。相反，高的CT值表示在没有增长，因为抗生素，显示易感性的有效运作，以测试的抗生素发生的样本。表1说明了确定的大肠杆菌的抗菌轮廓大肠杆菌和S.金黄色葡萄球菌菌株。所有CT值报告，并使用在协议文本中提到的公式（7.1），两个截止Ct值计算lated区分性和易感性。该菌株是耐抗生素，如果报告的CT值比计算截止Ct值（反之亦然）。 <img alt="图1" src="/files/ftp_upload/3254/3254fig1.jpg" /> 图1。使用实时16S rDNA的PCR技术的病原鉴定和药敏试验程序流程图。 <img alt="图2" src="/files/ftp_upload/3254/3254fig2.jpg" /> 图2鉴别法：血培养阳性扩增曲线和循环阈值（Ct值）由通用16S rDNA探针检测，而特异性探针因果病原体的鉴定。 A.血培养扩增曲线包含大肠杆菌大肠杆菌 ; B.血培养扩增曲线， 包含 S 金黄色葡萄球菌 ; Pseu AE，Pseudomon为绿脓杆菌; UNI，16S通用探针;大肠杆菌中， 大肠杆菌探头; Pseu SP， 假单胞菌 。探头; S.肺炎， 肺炎链球菌探头，链球菌属， 链球菌属。探头;肠杆菌， 肠球菌 。探头;金黄色葡萄球菌， 金黄色葡萄球菌探头;金黄色葡萄球菌属， 葡萄球菌属。探头。 <img alt="图3" src="/files/ftp_upload/3254/3254fig3.jpg" /> 图3扩增曲线的大肠杆菌药敏试验。 大肠杆菌分离 （样品1）。每条曲线代表该菌株培养的一种抗生素。一个早期信号，这意味着该菌株生长在测试抗生素存在，因此对抗生素耐药细菌高负荷引起的。后期的信号表明，该菌株已不存在抗生素生长，换句话说，很容易。 <table border="1"><tbody&gt; <tr><td> 示例1：E大肠杆菌 </td><td></td><td></td><td> 示例2：S金黄色葡萄球菌 </td><td></td><td></td></tr><tr><td> 助攻 </td><td> CT </td><td> R / S分析 </td><td> 助攻 </td><td> CT </td><td> R / S分析 </td></tr><tr><td>阿莫西林8毫克/升</td><td> 16,83 </td><td> ŕ </td><td>阿莫西林0.25毫克/升</td><td> 21,03 </td><td> ŕ </td></tr><tr><td>阿莫西林 - 克拉维8/4毫克/升</td><td> 17,36 </td><td> ŕ </td><td>苯唑2毫克/升</td><td> 25,80 </td><td> 小号 </td></tr><tr><td>哌拉西林16毫克/升</td><td> 16,67 </td><td> ŕ </td><td>万古霉素2毫克/升</td><td> 25,20 </td><td> 小号 </td></tr><tr><td>哌拉西林他唑巴坦16 /4毫克/升</td><td> 24,15 </td><td> 小号 </td><td>庆大霉素4毫克/升</td><td> 25,86 </td><td> 小号 </td></tr><tr><td>环丙沙星1毫克/升</td><td> 29,72 </td><td> 小号 </td><td>复方新诺明2/38毫克/升</td><td> 24,62 </td><td> 小号 </td></tr><tr><td>头孢他啶1毫克/升</td><td> 24,03 </td><td> 小号 </td><td></td><td></td><td></td></tr><tr><td>头孢他啶8毫克/升</td><td> 26,58 </td><td> 小号 </td><td></td><td></td><td></td></tr><tr><td>庆大霉素4毫克/升</td><td> 29,83 </td><td> 小号 </td><td></td><td></td><td></td></tr><tr><td>复方新诺明2/38毫克/升</td><td> 27,60 </td><td> 小号 </td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>负增长的控制（抗生素的混合物） </td><td> 30,41 </td><td></td><td>负增长的控制（样品保存于4°C） </td><td> 27,42 </td><td></td></tr><tr><td>正增长控制</td><td> 16,90 </td><td></td><td>正增长控制</td><td> 20,22 </td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>切断CT值1 * </td><td> 21,76 </td><td></td><td>截止CT值1 *** </td><td> 23,82 </td><td></td></tr><tr><td>切断CT值2 ** </td><td> 18,75 </td><td></td><td>停产关闭CT值2 **** </td><td> 22,02 </td><td></td></tr><tr><td> *阿莫西林，阿莫西林 - 克拉维酸，环丙沙星，gentamic复方新诺明 **哌拉西林，哌拉西林他唑巴坦，头孢他啶</td><td></td><td></td><td>万古霉素，庆大霉素*** ****对阿莫西林，苯唑西林和复方新诺明</td><td></td><td></td></tr></tbody></table> 表1。药敏试验的两个样本 （ 大肠杆菌和金黄色葡萄球菌 ）的测定 。的PCR检测CT值被复制到这个Excel文件，因为它可以自动计算出从正面和负面的生长控制的两个截止CT alues​​，使用在协议文本中的公式。如果抗生素比截止Ct值低的CT值，应变是耐抗生素，如果Ct值高于截止，应变是容易的。

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我们什么都没有透露。

这里描述的协议，使病原体的快速识别，并提供了一​​个功能抗菌的个人资料，可能会导致早期使用适当的抗生素，从而提高了患者的血液感染的预后。根据测试所要求的条件，即低成本，高吞吐量，最小的周转时间，测试条件下可以进行调整。整个过程可以在一个工作日内完成。此外，该协议的两个部分，可以同时进行，从而降低了周转时间显着。这里介绍，识别面板是一个在我们医院的临床相关细菌的选择。由于主要原理是针对16S基因区域，可以设计和其他微生物的特异性探针，加入检测。完整的检测最初用于快速分析的血培养，但也可以使用O处理进一步样品材料。这也是为药敏试验使用抗生素的情况下，可以添加更多或其他抗生素，根据当地的耐药模式和准则。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>氯化钠（NaCl） </td><td>默克化工技术</td><td> 106404 </td><td> 0.9％的水</td></tr><tr><td>真空采血管SST血清分离管5毫升</td><td>屋宇署诊断系统</td><td> 367986 </td><td></td></tr><tr><td>穆勒韩丁第二肉汤</td><td>屋宇署诊断Dystems </td><td> 212322 </td><td> 44克/在L水</td></tr><tr><td>离心Rotixa 50卢比</td><td>安德烈亚斯·海蒂诗GMBH＆CO KG </td><td> 4910 </td><td></td></tr><tr><td>离心5415ð </td><td> Eppendorf公司</td><td>已停产</td><td></td></tr><tr><td>引物</td> &lt;TD&gt; Sigma-Aldrich公司</td><td> NA </td><td></td></tr><tr><td>探头</td><td> Sigma-Aldrich/Applied生物系统公司</td><td> NA </td><td></td></tr><tr><td> TaqManEnvironmental主混合2.0 </td><td>应用生物系统公司</td><td> 4396838 </td><td></td></tr><tr><td> IQ SYBRGreen Supermix </td><td> BIO-RAD实验室BV公司</td><td> 170-8880 </td><td></td></tr><tr><td>微安光学96孔反应板</td><td>应用生物系统公司</td><td> N8010560 </td><td></td></tr><tr><td>微安光学胶膜</td><td>应用生物系统公司</td><td> 4311971 </td><td></td></tr><tr><td> iQ的96孔PCR板</td><td> BIO-RAD实验室BV公司</td><td> 223-9441 </td><td></td></tr><tr><td> microseal乙粘接密封</td><td> Bio-Rad公司劳动atories BV公司</td><td> MSB-1001 </td><td></td></tr><tr><td>实时PCR检测系统</td><td>应用生物系统公司</td><td> ABI PRISM 7900HT </td><td></td></tr><tr><td>实时PCR检测系统</td><td> BIO-RAD实验室BV公司</td><td> MyiQ™实时单色</td><td></td></tr></tbody></table>

one-day workflow scheme for bacterial pathogen detection and antimicrobial resistance testing from blood cultures

血液感染相关死亡率高，因为可能表现败血症，严重败血症和感染性休克1。因此，适当的抗生素治疗的快速管理是首要的重要性，在治疗血液感染。在这个过程中的关键要素是时机，在很大程度上依赖细菌鉴定和药敏试验的结果。这些参数都定期获得文化为基础的测试，这是耗费时间和平均24-48小时，2，4。这项研究的目的是开发基于DNA检测血液感染的快速识别，以及快速药敏试验。第一个实验是1真细菌16S RDNA-实时PCR检测与种属特异性探针5补充。使用这些探头，革兰氏阴性菌，包括假单胞菌，绿脓杆菌和大肠杆菌以及革兰氏阳性细菌，包括葡萄球菌，金黄色葡萄球菌，肠球菌，链球菌属，肺炎链球菌可以区别开来。使用这种多功能检测，致病微生物鉴定后2小时。 其次，我们开发了一个半分子检测与药敏试验金黄色葡萄球菌，肠球菌 。 （兼）需氧革兰氏阴性菌棒6。此法是基于在其中的PCR被用来测量7细菌生长的研究。直接取血培养的细菌培养与抗生素的选择6小时，1 SYBR绿色为基础的实时PCR检测后确定的生长抑制。这两种方法的结合，可以直接选择一个合适的抗生素治疗的同一天（ 图1）。结论分子，双方鉴定及药敏分析提供了一个更快的替代方案病原体检测，并能改善血液感染的诊断。

Watch this Scientific Journal Video about One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures at JoVE.com

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

病原菌诊断一个简单的为期一天的工作流程方案的设计，使血液感染的快速识别。列入的8个临床相关细菌的目标和其抗生素耐药谱提供的医生就在同一天最初的洞察力，从而导致更充分的治疗。

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. ...

one-day-workflow-scheme-for-bacterial-pathogen-detection

Research

JoVE Journal

Immunology and Infection

25.1K 次观看.  Maastricht University Medical Center. 病原菌诊断一个简单的为期一天的工作流程方案的设计，使血液感染的快速识别。列入的8个临床相关细菌的目标和其抗生素耐药谱提供的医生就在同一天最初的洞察力，从而导致更充分的治疗。

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

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

一个1.5小时的程序，鉴定肠球菌直接从血培养的物种

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

科研

免疫与感染

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

由细菌病原体入侵人体细胞

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in solution, has shown considerable potential in biochemical sensing applications with unprecedented sensitivity and specificity1-5. The drug-target interactions generate surface stress, causing the cantilever to bend, and the signal can be analyzed optically when it is illuminated by a laser. The change in surface stress measured with nano-scale precision allows disruptions of the biomechanics of model bacterial cell wall targets to be tracked in real time. Despite offering considerable advantages, multiple cantilever sensor arrays have never been applied in quantifying drug-target binding interactions.
Here, we report on the use of silicon multiple cantilever arrays coated with alkanethiol self-assembled monolayers mimicking bacterial cell wall matrix to quantitatively study antibiotic binding interactions. To understand the impact of vancomycin on the mechanics of bacterial cell wall structures1,6,7.&#160;We developed a new model1 which proposes that cantilever bending can be described by two independent factors; i) namely a chemical factor, which is given by a classical Langmuir adsorption isotherm, from which we calculate the thermodynamic equilibrium dissociation constant (Kd) and ii) a geometrical factor, essentially a measure of how bacterial peptide receptors are distributed on the cantilever surface. The surface distribution of peptide receptors (p) is used to investigate the dependence of geometry and ligand loading. It is shown that a threshold value of p ~10% is critical to sensing applications. Below which there is no detectable bending signal while above this value, the bending signal increases almost linearly,&#160;revealing that stress is a product of a local chemical binding factor and a geometrical factor combined by the mechanical connectivity of reacted regions and provides a new paradigm for design of powerful agents to combat superbug infections.

Acquired resistance to antibiotics is a major public healthcare problem and is presently ranked by the WHO as one of the greatest threats to human life. Here we describe the use of cantilever technology to quantify antibacterial resistance, critical to the discovery of novel and powerful agents against multidrug resistant bacteria.

纳米力学的药物靶标相互作用及耐药性检测

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in ...

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 ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

为快速细菌鉴定和药敏试验的准备血培养微丸

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

环介导等温扩增（LAMP）法进行的物种特异性检测<em&gt;艾美耳球虫</em&gt;可感染鸡

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.