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混合, 而涡流在 875 rpm。然后添加解决方案3。继续涡流60分钟. </li>
		<li> 离心20分钟, 在 1620 x g. 离心机上清液20分钟, 在 2880 x g. </li>
		<li> 通过透析净化终末清液。将上清液添加到透析袋 (分子6和 #8722; 8 K), 并将其放在含有磷酸盐缓冲盐水 (PBS) (pH = 7.4) 和自旋棒的烧杯中。让它浸泡12小时, 用新的缓冲器每2-3 小时更换一次. </li>
	</ol> </li> <li> IONPs 的目标特定抗体与表面的共轭 <ol> <li> 准备以下四解决方案: 溶液 A: 5 毫克 1-乙基 3-(3-dimethylaminopropyl) 二盐酸盐 (氯甲酸) 250 和 #181; L MES [2-( N -吗) 磺酸] 缓冲液 (0.1 米, pH = 6.8), 溶液 B: 3 毫克 n-羟基琥珀酰亚胺 (NHS) 在250和 #181; MES 缓冲器 (0.1 米, ph 值 = 6.8), 溶液 C: 5 和 #181; IgG1, 大肠杆菌单克隆, 225 和 #181; 磷酸缓冲盐水 (PBS) (ph 7.4), 溶液 D: 4 毫升的 IONP (5.0 摩尔) 在1毫升 PBS (pH 7.4). </li>
		<li> 将 2-(N-吗) 磺酸 (MES) 缓冲区添加到解决方案 a 中, 然后在十五年代内立即将解决方案 a 添加到解决方案 D 中, 并在每次增加混合后反转解决方案. </li>
		<li> 立即开始在三分钟内将解决方案 B 添加到解决方案 D 的小增量中。在每次添加后通过反转解决方案进行混合. </li>
		<li> 将解决方案 C 添加到解决方案中, 增量为5和 #181; L, 在每增加混合后反转解决方案. </li>
		<li> 允许反应在室温下持续3小时, 然后在晚上4和 #176 继续孵化. </li>
		<li> 使用 PBS (pH = 7.4, 最终 [Fe] = 3.5 摩尔) 将产生的 Ab 共轭 IONPs 通过磁性柱净化, 以去除任何游离抗体。
		<ol> <li> 用 PBS (1 毫升) 清洗磁性柱。将柱连接到磁板上, 并添加 IONP 溶液. </li>
			<li> 再用 PBS (1 毫升) 清洗磁性柱。从磁性板中取出磁性柱。将 PBS 添加到列中并收集解决方案。存储在4和 #176; C </li> </ol> </li> </ol> </li> <li> 封装荧光11和 #39;-Dioctadecyl-3,3,3 和 #39;, 3 和 #39;-Tetramethylindocarbocyanine 高氯酸盐 (DiI) 染料的聚丙烯酸 (临机) 涂层的抗体共轭 IONPs 使用溶剂扩散法。
	<ol> <li> 到4毫升的 IONPs, 添加滴2.0 和 #181; DiI 染料 (2 摩尔) 在100和 #181 中的 l, 以连续混合在 1100 rpm 的亚砜 l. </li>
		<li> 透析结果解决方案 (分子 6-8 KDa, 12 h) 对抗 PBS, 创建最终的 MFnS 解决方案, 最终铁浓度为 [Fe] = 2 摩尔. </li>
	</ol> </li> <li> 特性 MFnS <ol> <li> 进行分光光度分析, 使用平板阅读器检测 595 nM 的封装 DiI 染料和荧光发射。
		<li> 动态光散射 <ol> <li> 将 MFnS 溶液的样品放到 zetasizer 中, 以确定 MFnS 的平均尺寸和表面电荷. 
			注意: 在这里, 功能 MFnS 的平均大小和表面电荷分别被发现为 77.09 nm 和-22.3 mV. </li>
		</ol> </li> </ol> </li> </ol> 2。细菌培养和砧木溶液制备 <ol> <li> 细菌培养 <ol> <li> 水合 freeze-dried 颗粒 ( 大肠杆菌 O157:H7) 在1毫升的营养液中, 然后再加入5毫升的肉汤。
		<li> 应用100和 #181; 此解决方案的 L 使用无菌接种循环, 然后在37和 #176 中孵化; C 为 24 h. </li>
		<li> 24 小时后, 从琼脂板中选择一个孤立的菌落, 并将其添加到15毫升的培养液中。在37和 #176 孵育, C 为4-6 小时, 同时监测光学密度值 (吸光度在 600 nm). </li>
		<li> 一旦获得0.1 的光学密度值, 停止培养, 并执行串行稀释. </li>
	</ol> </li> <li> 串行稀释 <ol> <li> 添加900和 #181; L 营养液为八无菌离心管。
		<li> 添加100和 #181; 细菌悬浮的 L 对第一个管子 (稀释 10 -1 ). </li>
		<li> 取100和 #181; L 从第一个管, 并添加它到第二个, 获得稀释 10 -2 。继续此模式的其余管, 最后稀释 10 -8 . </li>
		<li> 传输100和 #181; 从每个试管中分离琼脂板, 并在37和 #176 孵育24小时; C. </li>
		<li> 第二天, 计算每个板块上的菌落形成单位 (CFUs)。选择 100 CFUs 的板材。使用相应的稀释进行进一步的实验 (100 和 #181; L = ~ 100 CFUs). 
		注意: 这里是 10 -6 稀释, 导致 100 CFUs. </li>
	</ol> </li> </ol> 3。利用 MFnS 快速检测大肠杆菌 O157:H7 <ol> <li> 使用各种 PBS 解决方案 (1X、pH 值7.4、300和 #181; L), 10 -6 细菌的数量增加, 导致 CFU 范围从1-100。在这些解决方案中添加一致的 MFnS (100 和 #181; L). </li>
	<li> 创建一个基线解决方案, 其中仅包含 PBS (1X、pH 7.4、300和 #181; l) 和 MFnS (100 和 #181; l). </li>
	<li> 在37和 #176 孵育溶液30分钟, 然后让它们冷却到室温. </li>
	<li> 将单个解决方案转移到磁 relaxometer (0.47 泰斯拉), 并在每个解决方案中的 CFUs 中记录弛豫时间 (T2) 的变化。
	<ol> <li> 若要记录 T2 值的更改, 请首先测量包含 PBS 和 MFnS 的基线解决方案的 T2 值。
		<ol> <li> 将解决方案和 #160 放置在磁性 relaxometer 中。打开相应的软件, 选择和 #34; T2 放松和 #34; 设置和按下 #34; 测量. #34; </li> </ol> </li> <li> 在基线 T2 的集合之后, 测量附加解决方案的 T2 值, 这些解是用各种细菌的浓度. 
		注: T2 的变化相当于从尖刺 T2 中减去的基线 T2. </li>
	</ol> </li> <li> 从磁性 relaxometer 中取出样品, 并将试管离心 2880 x g, 10 分钟. </li>
	<li> 醒酒100和 #181 中的上清和重细菌颗粒; PBS (1X, pH 7.4). </li>
	<li> 添加80和 #181; 每个悬浮为96个井板, 记录荧光强度在 595 nM. 
	注: 测试可以重复使用不同的溶剂, 包括湖泊水, 牛奶和其他如在结果部分所述 </li> </ol>

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这一纳米技术的示范应用尚未得到 FDA 的批准。

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。

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		<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 ...

foodborne-pathogen-screening-using-magneto-fluorescent-nanosensor

Research

JoVE Journal

Immunology and Infection

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

7.6K 次观看.  Pittsburg State University. 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。

视频: 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

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

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

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

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

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

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

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

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

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

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of 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

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 new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

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

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 ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

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

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 outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

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

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

使用细菌共轭的 Enterobacterial 菌株 (如大肠杆菌或志贺菌福氏) 创建稠密的转插入库

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

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

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.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

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

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

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 ...

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.