Name: rapid identification of gram negative bacteria from blood culture broth using maldi-tof mass spectrometry
Uploaded: 2014-05-28T13:45:00
Description: Watch this Scientific Journal Video about Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry at JoVE.com

<html> 1，血培养液标为“正” <ol><li>从连续监测温育箱中取出信号血培养瓶，并把它变成一个生物安全柜。注意：瓶可以含有有害微生物和普遍预防需要遵循。由于采样传染性气溶胶的危险性，所有抽样程序必须在生物安全II级层流柜中进行。 </li></ol> 2，革兰氏染色准备<ol><li>从信号血培养肉汤按当地机构的协议准备革兰氏染色。注意：当革兰氏阴性生物体的显微鉴定的血培?...

<ol>
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Dis. 32 (5), 699-704 (2012).</li><li>Christner, M., Rohde, H., Wolters, M., Sobottka, I., Wegscheider, K., Aepfelbacher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+of+bacteria+from+positive+blood+culture+bottles+by+use+of+matrix-assisted+laser+desorption-ionization+time+of+flight+mass+spectrometry+fingerprinting.">Rapid identification of bacteria from positive blood culture bottles by use of matrix-assisted laser desorption-ionization time of flight mass spectrometry fingerprinting.</a> J. Clin. Microbiol. 48 (5), 1584-1591 (2010).</li><li>Gray, T. J., Thomas, L., Olma, T., Iredell, J. R., Chen, S. 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Med. Microbiol. 62 (2), 223-2231 (2013).</li><li>Saffert, R. T., Cunningham, S. A., Mandrekar, J., Patel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+three+preparatory+methods+for+detection+of+bacteremia+by+MALDI-TOF+mass+spectrometry.">Comparison of three preparatory methods for detection of bacteremia by MALDI-TOF mass spectrometry.</a> Diagn. Microbiol. Infect. Dis. 73 (1), 21-26 (2012).</li><li>Consoir, C., L&#246;rch, D., Schneider, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+identification+of+bacteria+from+charcoal-containing+blood+culture+bottles+using+matrix-assisted+laser+desorption/ionisation+time-of-flight+mass+spectrometry.">Direct identification of bacteria from charcoal-containing blood culture bottles using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.</a> Eur. J. Clin. Microbiol. Infect. Dis. 31 (10), 2843-2850 (2012).</li><li>Martiny, D., Dediste, A., Vandenberg, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+an+in-house+method+and+the+commercial+Sepsityper&#8482;+kit+for+bacterial+identification+directly+from+positive+blood+culture+broths+by+matrix-assisted+laser+desorption-ionisation+time-of-flight+mass+spectrometry.">Comparison of an in-house method and the commercial Sepsityper&#8482; kit for bacterial identification directly from positive blood culture broths by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry.</a> Eur. J. Clin. Microbiol. Infect. Dis. 31 (9), 2269-2281 (2012).</li><li>Moussaoui, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-assisted+laser+desorption+ionization+time-of-flight+mass+spectrometry+identifies+90%+of+bacteria+directly+from+blood+culture+vials.">Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identifies 90% of bacteria directly from blood culture vials.</a> Clin Microbiol Infect. 16 (11), 1631-1638 (2010).</li><li>Novel, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=improved+sample+preparation+for+rapid,+direct+identification+from+positive+blood+cultures+using+matrix-assisted+laser+desorption/ionization+time-of-flight+(MALDI-TOF)+mass+spectrometry.">improved sample preparation for rapid, direct identification from positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.</a> J. Mol. Diagn. 13 (6), 701-706 (2011).</li><li>Fothergill, A., Kasinathan, V., Hyman, J., Walsh, J., Drake, T., Wang, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Identification+of+Bacteria+and+Yeasts+from+Positive-Blood-Culture+Bottles+by+Using+a+Lysis-Filtration+Method+and+Matrix-Assisted+Laser+Desorption+Ionization-Time+of+Flight+Mass+Spectrum+Analysis+with+the+SARAMIS+Database.">Rapid Identification of Bacteria and Yeasts from Positive-Blood-Culture Bottles by Using a Lysis-Filtration Method and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrum Analysis with the SARAMIS Database.</a> J. Clin. Microbiol. 51 (3), 805-809 (2013).</li></ol>

运用MALDI-TOF MS到后离心步骤都与足够的照顾不进行重新混合分离的成分血培养肉汤时，这一点很重要。特别重要的是，除去血培养成分和人类细胞的蛋白质，包括血红蛋白，这可能会产生尖峰与MALDI-TOF光谱干扰。 虽然MS制造商推荐切得分≥2.0的物种和≥1.7，属鉴定，其他报告表明较低的对数得分（范围≥1.4至≥1.6）时，适用于公元前肉汤10,12,14-18可以实现。在临床微?...

由于细菌患者的血流感染（BSI）继续有较高的住院死亡率，范围从6-48％1。适当的经验性抗生素交付促进生存和在严重脓毒症患者的子集，每个小时的延迟，以适当的治疗相关的生存2,3下降。因此，临床实验室的主要目标是快速检测，识别和沟通细菌在血培养存在的，告知临床决策。它已经证明，微生物实验室对抗菌治疗在报告的革兰氏染色4，最近的一次影响最大，观察研究证明，飞行质谱基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）直接在血培养液进行影响超过三分之一BSI引起的革兰阴性菌5订明。 ，6,7微生物的鉴定。该技术是目前公认，并已集成到许多实验室进行快速准确的识别微生物中分离固体培养基上6,8。直接应用MALDI-TOF MS对血培养（BC）的肉汤已经表示“积极”的微生物，因为获得低成本的早期识别微生物的潜在的临床医生和实验室管理人员的上诉。 直接应用MALDI-TOF MS的血培养液的临床应用已被广泛观察到的灵敏度，当与鉴定标准表型文化为基础的方法，以成功识别革兰阴性菌不等的报告相比，限制从47-98.9 ％9-11。在灵敏度的变化可能涉及对BC肉汤组成，初始细菌浓度，变化的样品制备方法，以及在研究人群9中遇到的革兰氏阴性生物体的数组。与这些其他的发布的协议相比，这里提出的方法可避免使用乙醇，氯化铵或附加的（非矩阵）乙腈。其结果是将细菌沉淀将保持存活（直到蛋白提取的点），允许潜在敏感性表型试验方法，可直接应用于这些微生物在液体培养基。此外，所提出的方法已经被证明是便宜的，可靠的，快速，细菌鉴定内的血培养物革兰氏染色结果25分钟内可用的，以最小的手上的时间12。 这种方法是利用甲酸提取一个简单的内部自旋裂解方案直接应用到血培养阳性肉汤识别革兰氏阴性bacteria与MALDI-TOF MS技术。

<table border="0" cellpadding="0" cellspacing="0" width="1319">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">BACTEC Plus Aerobic/F Medium</td>
			<td style="width:307px;">Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right" style="width:188px;">442192</td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BACTEC Lytic/10 Anaerobic /F Medium</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">442265</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BACTEC Peds Plus Medium</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">442194</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer - Blood transfer device</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">364880</td>
			<td>Single use sampling device reducing the risk of needlestick injury</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer SST 5.0mL, Advance plus</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">367954</td>
			<td>Serum separating tube</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe 10 mL</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">302143</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette</td>
			<td>Samco, USA</td>
			<td>222-20S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette (fine tipped)</td>
			<td>Samco, USA</td>
			<td>232-20S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcentrifuge tube (2.0 mL)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>0030.120.094</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile water (DNAse and RNAse free)</td>
			<td>Life Technologies, Carlsbad, California, USA</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>F0507</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrix solution&#160;</td>
			<td>Bruker Daltonics, Bremen Germany</td>
			<td align="right">285074</td>
			<td>10 mg/ml &#945;-cyano-4-hydroxycinnamic acid, 50% acetonitrile, 2.5% trifluoroacetic acid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Benchtop microflex LT MALDI-TOF MS</td>
			<td>Bruker Daltonics, Bremen Germany</td>
			<td></td>
			<td>Utilizing BioTyper 3.1 (Build 65) and FlexControl 3.3 (Build 99) software</td>
		</tr>
	</tbody>
</table>

rapid identification of gram negative bacteria from blood culture broth using maldi-tof mass spectrometry

临床微生物学实验室的一个重要作用是提供快速鉴定细菌，导致血液感染的。传统的识别需要信号血培养肉汤识别可用的亚文化不仅已经成熟后，在固体琼脂菌落。 MALDI-TOF MS是用于识别大多数临床相关的细菌时，适用于菌落在固体培养基上可靠的，快速的方法。 MALDI-TOF MS直接到血培养液中的应用是一个有吸引力的方法，因为它有潜力加速了细菌的种属鉴定，提高临床管理。然而，要克服的一个重要问题是，在分析前除去包含在血培养标本，如不去除，干扰MS谱，并可能导致判别识别分数不足或低干扰的树脂，蛋白质和血红蛋白。此外，有必要集中硝化细菌IA开发足够的质量谱。该方法描述了浓缩，纯化和提取的革兰氏阴性细菌，允许从一个信号血液培养液中的早期识别细菌。

所生成的MALDI-TOF MS谱进行比较，以光谱的集成参考数据库。对数分数分配给测试分离和参考数据库隔离之间的比赛的信心，用分数≥1.7所需的鉴定可能要属水平（ 图1），≥2.0为可能的识别物种（ 图的建议2）。报告以种的水平时，得分≥1.7和所述第一5标识所匹配的打字软件是一致的（ 图1）12。图3演示了当混合物种的血培养液进行分析?...

Watch this Scientific Journal Video about Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry at JoVE.com

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

的飞行（MALDI-TOF）质谱（MS），直接向血培养肉汤基质辅助激光解吸/电离飞行时间质谱的应用加快细菌的鉴定。该方法是一种快速和可靠的方法，可直接从血培养肉汤鉴定革兰氏阴性细菌。

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 ...

Research

JoVE Journal

Immunology and Infection

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

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

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

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

热酚水提取纯化及革兰氏阴性菌脂多糖的可视化

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Detection of Bacteria Using Fluorogenic DNAzymes

检测使用荧光脱氧核酶的细菌

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

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

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 ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

Isolation of Double Negative ÃƒÅ½Ã‚Â±ÃƒÅ½Ã‚Â² T Cells from the Kidney

从肾脏双重否定αβ性T细胞的分离

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

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

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 ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

参考微量稀释法达巴万星<em&gt;体外</em&gt;细菌敏感性测试生长有氧

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

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

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 ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

巨噬细胞集落刺激因子分化的人单核细胞衍生的巨噬细胞的培养

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...