Name: foodborne pathogen screening using magneto-fluorescent nanosensor: rapid detection of e. coli o157:h7
Uploaded: 2017-09-17T14:00:00
Description: Watch this Scientific Journal Video about Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7 at JoVE.com

这项工作是支持 INBRE P20GM103418, 堪萨斯大豆委员会 (肯尼迪航天公司/电源 1663), ACS 脉冲 56629-UNI7 和电源聚合物化学启动基金, 所有的 SS。我们感谢大学摄影师, Mr. 雅各 Anselmi, 为他出色的视频工作。我们还感谢 Mr. 罗杰 Heckert 和 Mrs. 卡塔奥义 Heckert 对研究的慷慨支持。

1.Synthesis 多参量磁荧光纳米 (MFnS) 的功能化. 
<ol> <li> 合成顺氧化铁纳米粒子 (IONPs) <ol> <li> 准备 IONP 合成, 准备以下3解决方案: 解决方案 1: FeCl 3 (0.70 g) 和 FeCl 2 在 H 2 O (2 毫升), 解决方案 2: NH 4 OH (2.0 毫升, 13.4 米) 在 h 2 O (15 毫升) 和解决方案 3: 聚丙烯酸 (0.855 g) 在 h 2 O (5 毫升)。
		<li> 将90和 #181; 2 米盐酸 (HCl) 添加到溶液 1, 然后立即与溶液2...

<ol>
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本协议的设计目的是尽可能简单地生成完全功能的 MFnS。但是, 根据用户的最终目标, 更改协议可能有用的许多关键点。例如, 使用不同的抗体可以使许多其他病原体成为靶向。此外, 本议定书不限于使用抗体作为靶分子。任何对目标病原体有特定约束力的分子, 如宿主细胞受体, 也可用作靶分子。只要靶向分子有一个主要胺或酒精组, 或可以功能化有一个, 它可能被共轭到表?...

在商业生产的食物和水源中, 细菌污染的持续发生, 已使人们对越来越快速和具体的诊断平台产生了需求。1,2一些更常见的细菌污染物是由沙门氏菌、葡萄球菌、李斯特菌、弧菌、志贺氏杆菌和大肠杆菌引起的。3,4这些病原体的细菌污染通常会导致发烧、霍乱、肠胃炎和腹泻等症状。4水资源的污染往往对社区造成严重和不利的影响, 无法获得足够的过滤水, 食品污染导致大量疾病和产品召回努力。5,6
为了减少因细菌污染引起的疾病的发生, 已经作出了许多努力, 以制定方法, 在销售或消费之前, 可以有效地对水和食物进行扫描。3技术, 如 PCR、1、7、8、9、10 ELISA、11、12循环介导的等温放大 (灯),13,14 ,15,16,17,18,19,20,21, 22、23、24最近被用于检测各种病原体。与传统的细菌培养方法相比, 这些技术在特异性和时间方面更有效率。然而, 这些技术仍在与虚假的正面和负面、复杂的程序和成本斗争。1,3,25正是由于这个原因, 多磁荧光纳米 (MFnS) 被提出作为细菌检测的替代方法。
这些纳米独特的配对磁松弛和荧光模式, 允许双检测平台, 既快速又准确。使用大肠杆菌O157:H7 作为样品的污染物, MFnS 在几分钟内检测到 1 CFU 的能力被证实。病原体特异抗体用于增加特异性, 并且磁性和荧光方式的组合允许检测和定量细菌污染物在两个低 high-contamination 范围。16在细菌污染的情况下, 由于特定病原体抗体的靶向能力, 纳米将围绕细菌群。磁性纳米和细菌之间的结合限制了磁性铁芯与周围的水质子之间的相互作用。这会导致 T2 松弛时间的增加, 如磁 relaxometer 所记录的那样。随着溶液中细菌浓度的升高, 纳米随细菌数量的增加而分散, 导致 T2 值降低。反之, 由于纳米直接与病原体的数量增加, 荧光辐射与细菌浓度的比例会增加。离心的样品, 和分离的细菌颗粒, 将只保存纳米颗粒直接附着在细菌, 消除任何自由漂浮的纳米, 并直接关联的荧光辐射与数量溶液中存在细菌。此机制的示意图表示形式在图 1中表示。
这个 MFnS 平台已经设计了点的筛选, 导致低成本和便携的特点。MFnS 在室温下是稳定的, 只有在非常低的浓度下才能准确检测细菌污染物。此外, 在合成后, 使用 MFnS 是简单的, 不需要在实地使用训练有素的专业人员。最后, 这个诊断平台允许高度可定制的目标, 提供一种手段, 这一平台可以用来检测各种病原体, 在许多不同的设置。

<table border="0" cellpadding="0" cellspacing="0" width="821">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ferrous Chloride Tetrahydrate</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">I90-500</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ferric Chloride Hexahydrate</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td>I88-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ammonium Hydroxide</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">A669S-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Hydrochloric Acid</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">A144S-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Polyacryllic Acid</td>
			<td style="width:156px;">Sigma-Aldrich</td>
			<td style="width:197px;">323667-100G</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">EDC</td>
			<td style="width:156px;">Thermofisher Scientific</td>
			<td style="width:197px;">22980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">NHS</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td>AC157270250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-E. coli O111 antibody&#160;</td>
			<td style="width:156px;">sera care</td>
			<td>5310-0352</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-E. coli O157:H7 antibody [P3C6]&#160;</td>
			<td style="width:156px;">Abcam</td>
			<td style="width:197px;">ab75244</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">DiI Stain</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">D282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Nutrient Broth</td>
			<td style="width:156px;">Difco</td>
			<td style="width:197px;">233000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Freeze-dried E. coli O157:H7 pellet</td>
			<td style="width:156px;">ATCC</td>
			<td style="width:197px;">700728</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Magnetic Relaxomteter&#160;</td>
			<td style="width:156px;">Bruker</td>
			<td style="width:197px;">mq20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Zetasizer</td>
			<td style="width:156px;">Malvern</td>
			<td style="width:197px;">NANO-ZS90</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Plate Reader&#160;</td>
			<td style="width:156px;">Tecan</td>
			<td style="width:197px;">Infinite M200 PRO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Magnetic Column&#160;</td>
			<td style="width:156px;">QuadroMACS</td>
			<td style="width:197px;">130-090-976</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Centrifuge</td>
			<td style="width:156px;">Eppendorf</td>
			<td style="width:197px;">5804 Series</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Centrifuge (accuSpin Micro 17)</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">13-100-676</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Floor Model Shaking Incubator</td>
			<td style="width:156px;">SHEL LAB</td>
			<td style="width:197px;">SSI5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Analytical Balance</td>
			<td style="width:156px;">Metler Toledo</td>
			<td style="width:197px;">ME104E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Digital Vortex Mixer</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">02-215-370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Open-Air Rocking Shaker</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">02-217-765</td>
			<td></td>
		</tr>
	</tbody>
</table>

foodborne pathogen screening using magneto-fluorescent nanosensor: rapid detection of e. coli o157:h7

出大肠杆菌O157:H7 已与水和食源性疾病联系在一起, 尽管目前使用的食品和 water-screening 方法仍然存在威胁。虽然传统的细菌检测方法, 如聚合酶链反应 (PCR) 和酶联免疫吸附试验 (ELISA) 可以明确检测致病污染物, 他们需要广泛的样本准备和漫长的等待期。此外, 这些做法需要先进的实验室仪器和设置, 必须由训练有素的专业人员执行。在此, 提出了一种简单的诊断技术的协议, 其特点是磁性和荧光参数在纳米平台的独特组合。所提出的多磁荧光纳米 (MFnS) 可以检测到在小于1小时内, 在溶液中存在的1菌落形成单元的大肠杆菌O157:H7 污染。此外, 还证实了 MFnS 在诸如牛奶和湖水等复杂介质中保持高度功能的能力。另外的特异性化验也被用来证明 MFnS 只检测特定目标细菌的能力, 即使在存在类似的细菌种类的情况下。磁性和荧光模式的配对允许检测和定量的病原体污染在广泛的浓度, 表现出其在早期和晚期污染检测的高性能。MFnS 的有效性、可承受性和可移植性使他们成为点筛选各种环境中的细菌污染物的理想候选者, 从水生水库到商业包装食品。

MFnS 操作的机制在图 1中表示。细菌污染物表面周围 MFnS 的聚类干扰了 MFnS 和周围氢核的磁性核心之间的相互作用。由于这种聚类, 磁松弛值增加。随着细菌污染物浓度的增加, 聚类减少, T2 值的变化减小。因此, 增加一个荧光形态是至关重要的。随着细菌浓度的增加, MFnS 所产生的荧光信号的强度增加, 从而使得对低浓度和高浓缩范围内细菌污染物的敏感检测成为可以。
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Watch this Scientific Journal Video about Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7 at JoVE.com

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

本协议的总体目标是通过磁性松弛和荧光发射方式的结合, 合成功能性纳米, 用于便携式、cost-effective 和快速检测特定靶向致病菌。

利用磁荧光纳米进行食源性致病菌筛选: 快速检测大肠杆菌O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

Research

JoVE Journal

Immunology and Infection

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

科研

免疫与感染

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

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. ...

Following Cell-fate in E. coli After Infection by Phage Lambda

Following Cell-fate in E. coli After Infection by Phage Lambda

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

活细胞视频显微镜病病原菌的吞噬作用

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

HIV逆转录酶和整合酶抑制剂的快速筛选

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

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

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

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 ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

快速分子检测和流感病毒A和B的分化

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...