Name: multiplexed isothermal amplification based diagnostic platform to detect zika, chikungunya, and dengue 1
Uploaded: 2018-03-13T13:30:00
Description: Watch this Scientific Journal Video about Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1 at JoVE.com

这项工作部分由 FDOH-7ZK15 和 NIAID 1R21AI128188-01 支助。该出版物中报告的研究部分由国家过敏和传染性疾病研究所支持, 部分由佛罗里达卫生部的生物医学研究计划提供。内容完全是作者的责任, 不一定代表 NIH 或佛罗里达卫生部的官方意见。动态组合化学有限责任公司对该项目的支持和贡献表示认可。
1登革热病毒 (应变 BOL-KW010) 是由佛罗里达州卫生局实验室提供的。疾病控制和预防中心慷慨地提供了 Zika 病毒和亚洲基孔肯亚病毒血统。基孔肯亚病毒的印度洋血统是由罗伯特. Tesh (世界新兴病毒和虫媒病毒中心) 提供的, 通过得克萨斯州加尔维斯顿的德克萨斯大学医学分支机构向超滤-FMEL。我们感谢贝拉米, b. 伊斯特蒙, 贝莱斯, k. 威金斯, R. ZimLer 和 k. Zirbel 协助感染研究。我们还感谢金硕士提供了 Q 纸。

注意: 蚊子是这项研究中唯一直接使用的动物。管理鸡的程序, 其血液被用来喂养受感染的蚊子, 被批准为 IACUC 协议 #201507682 由佛罗里达大学机构动物护理和使用委员会. 病毒传播和蚊虫感染研究是在佛罗里达州的维罗海滩医学昆虫实验室的 BSL-3 设施进行的 RT 灯实验在 FfAME 和国产生物分子科学有限责任公司在阿拉丘瓦, 佛罗里达州共享的 BSL-2 实验室进行。
1. 引物和绞线置换探?...

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包括 Zika、基孔肯亚和登革热在内的蚊虫传播病毒威胁到公共卫生, 最近的 Zika 暴发突出表明需要低成本的护理点检测替代品来进行患者诊断, 以及进行蚊虫监测。等温放大方法是作为可负担的替代 PCR 系统开发的。特别是, 基于 RT 灯的平台已被用于检测范围广泛的病原体。然而, 等温平台的使用主要限于单目标检测。这里报告的方法使用一个改进的 RT 灯允许同时检测三个不同的目标在一个单一的?...

蚊虫传播的病毒感染, 包括登革热、基孔肯亚和 Zika 病毒正在上升, 需要立即管理战略。登革热和基孔肯亚病毒已经在许多热带地区流行, 在那里 Zika 现在在西半球传播1。Zika 病毒, 如登革热, 是Flaviviridae家族的成员, 原产于非洲, 有一个亚洲和两个非洲遗传血统2。尽管 Zika 病毒的鉴定可以追溯到 1947, 但在太平洋和美洲出现前半个世纪, 人类的 Zika 感染仍然是零星的。第一次报告说, 2007年在密克罗尼西亚联邦发生的 Zika 热爆发, 随后是在2013年和2014法属波利尼西亚。美洲第一次大规模暴发发生在2015年巴西。
Zika、基孔肯亚和登革热病毒主要由伊蚊蚊和白纹伊蚊传播。然而, Zika 有额外的下游人到人传输的可能性, 可能通过性接触传播, 母婴互动, 并通过母乳喂养3,4,5。Zika 发烧最初被认为只导致轻度疾病。然而, 它后来与成人的格林-巴利综合征, 新生儿小头和慢性肌肉骨骼疾病, 可能持续数月至数年。诊断 Zika 疾病可能是挑战, 因为 Zika 感染的症状类似于其他蚊子传播病毒6。这些病毒的常见联合感染使鉴别诊断更加具有挑战性7,8。因此, 需要对 Zika 和其他病毒的核酸进行快速、可靠的检测, 以便实时了解流行病学, 启动控制和预防措施, 并管理病人护理9。
目前对这些病毒的诊断测试包括血清学测试、病毒分离、病毒测序和反向转录 pcr (rt-pcr)。标准血清学方法往往受到不充分的敏感性, 结果可能是复杂的交叉反应的病人谁曾感染其他 flaviviruses。
因此, 核酸检测仍然是检测和鉴别这些病毒最可靠的方法。Zika 和其他蚊虫传播病毒的检测通常是使用 rt-pcr 或实时 rt-pcr 在各种生物液中进行, 如血清、尿液、唾液、精子、母乳和脑液10,11。尿液和唾液样本通常比血液更可取, 因为它们的 PCR 抑制较少, 病毒负载更高, 病毒存在时间较长, 而且收集和处理的易用性提高了12,13。然而, 基于 RT PCR 的诊断测试包括大量的样品准备步骤和昂贵的热循环设备, 使其对护理点的优化不那么理想。
反向转录环介导的等温放大 (rt 灯) 已成为一个强大的 rt-pcr 替代方案, 由于其高灵敏度和特异性14, 它对生物样品中的抑制物质的容忍15, 和单一温度下的操作, 大大降低了检测的复杂性和相关成本, 使之适合低资源环境。RT 灯, 因为它是经典的实施, 包括六引物, 绑定到八个不同的区域内的目标 RNA。它运行在恒定的温度在60°c 和70°c 之间, 并且使用一条反向逆转录酶和一根有强的链置换活动的脱氧核糖核酸 pcr。
在 RT 灯的初始阶段, 向前和向后内部引物 (FIP 和保险,图 1A) 连同外向前和向后引物 (F3 和 B3) 形成哑铃结构, 种子结构的指数灯放大。放大是进一步加速的循环向前和向后引物 (LF 和磅), 这是设计来绑定的单一搁浅地区的哑铃, 并导致形成 concatemers 与多个重复循环16。基于浊度或锂染料读数的经典光源检测方法并不完全适合 Zika 的点检, 在那里需要某种程度的多路复用17,18,19。在这些系统中, 多路复用不容易获得, 因为由于目标放大, 它们容易产生误报。
为了管理这些问题, 文献以 "线置换探针" 的形式向经典的 RT 灯体系结构20、21、22添加了一个附加组件。每个探针都有一个序列特定的双链区域和一个单链启动区域。该探针与单绞区被标记为 5 '-荧光, 互补探针是修改与 3 '-端淬火。在没有目标的情况下, 由于互补探针链的杂交, 没有发现荧光, 从而使荧光和淬火接近。在目标存在的情况下, 荧光探针的单链部分与靶上的补体相结合, 然后通过链置换聚合酶进行扩展。进一步的聚合酶引伸反向引物导致分离的淬火链从其互补荧光标记链, 允许发射荧光(图 1B)。通过这种设计, 在哑铃形成后产生信号, 减少了假阳性信号的几率。
链置换探针的双绞线可以是任意序列, 当应用多路复用时, 相同的序列可以与不同的荧光-淬火对一起使用。通过这种体系结构, 直接将病毒污染的尿液、血清或蚊子样本压扁在纸上, 而不进行样品制备。在 30-45 分钟内产生肉眼可见的三色荧光读出, 并使用蓝色 LED 和橙色过滤器在3维打印的观察框中可视化信号。冷冻干燥的 RT 灯试剂启用此工具包的部署, 以降低资源设置, 不需要制冷。

<table>
	<tbody>
		<tr>
			<td>SafeBlue Illuminator/ Electrophoresis System, MBE-150-PLUS</td>
			<td>Major Science</td>
			<td>MBE-150</td>
			<td>Gel electrophoresis</td>
		</tr>
		<tr>
			<td>G:BOX F3</td>
			<td>Syngene</td>
			<td>G:BOX F3</td>
			<td>Gel imaging</td>
		</tr>
		<tr>
			<td>LightCycler 480 Instrument II, 96-well</td>
			<td>Roche Applied Science</td>
			<td>05 015 278 001</td>
			<td>Real-time PCR</td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane</td>
			<td>Millipore Sigma</td>
			<td>UFC501096</td>
			<td>ultrafiltraton membrane for dialysis</td>
		</tr>
		<tr>
			<td>Eppendorf 5417C Centrifuge</td>
			<td>Marshall Scientific</td>
			<td>EP-5417C</td>
			<td>centrifuge</td>
		</tr>
		<tr>
			<td>Myblock Mini Drybath</td>
			<td>Benchmark Scientific</td>
			<td>BSH200</td>
			<td>drybath</td>
		</tr>
		<tr>
			<td>FreeZone Plus 6 Liter Cascade Console Freeze Dry System</td>
			<td>Labconco</td>
			<td>7934020</td>
			<td>lyophilizer</td>
		</tr>
		<tr>
			<td>all priers and probes</td>
			<td>IDT</td>
			<td>custom</td>
			<td>RT-LAMP primers and probes</td>
		</tr>
		<tr>
			<td>dNTP set</td>
			<td>Bioline</td>
			<td>BIO-39049</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyuridine Triphosphate (dUTP)</td>
			<td>Promega</td>
			<td>U1191</td>
			<td></td>
		</tr>
		<tr>
			<td>Bst&#160;2.0 WarmStart&#160;DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0538L</td>
			<td>enzyme</td>
		</tr>
		<tr>
			<td>WarmStart RTx Reverse Transcriptase</td>
			<td>New England Biolabs</td>
			<td>M0380L</td>
			<td>enzyme</td>
		</tr>
		<tr>
			<td>RNase Inhibitor, Murine</td>
			<td>New England Biolabs</td>
			<td>M0314L</td>
			<td>enzyme</td>
		</tr>
		<tr>
			<td>Antarctic Thermolabile UDG</td>
			<td>New England Biolabs</td>
			<td>M0372L</td>
			<td>enzyme</td>
		</tr>
		<tr>
			<td>50bp DNA Step Ladder</td>
			<td>Promega</td>
			<td>G4521</td>
			<td>marker</td>
		</tr>
		<tr>
			<td>LightCycler 480 Multiwell Plate 96, white</td>
			<td>Roche Applied Science</td>
			<td>4729692001</td>
			<td>real-time RT-LAMP analysis</td>
		</tr>
	</tbody>
</table>

multiplexed isothermal amplification based diagnostic platform to detect zika, chikungunya, and dengue 1

Zika、登革热和基孔肯亚病毒是由蚊子传播的, 引起类似患者症状的疾病。然而, 他们有不同的下游病人对病人的传输潜能, 并需要非常不同的病人治疗。因此, 最近的 Zika 暴发使人们迫切需要开发快速鉴别这些病毒的患者和被困蚊子的工具, 选择正确的病人治疗, 并实时了解和管理他们的流行病学。
不幸的是, 目前的诊断测试, 包括那些收到2016紧急使用授权和快速轨道状态, 检测病毒 RNA 的反向转录聚合酶链反应 (RT PCR), 这需要检测, 训练有素的用户, 并大量的样品准备。因此, 他们必须被送往 "批准" 的参考实验室, 需要时间。事实上, 在 2016年8月, 疾病控制中心 (CDC) 正在询问那些被蚊子叮咬的孕妇, 并在得知他们是否感染之前, 在2周的时间里, Zika 的皮疹来等待一个不可接受的4星期。我们非常需要测试, 可以在现场进行, 很少的资源, 并由训练有素但不一定是授权的人员。
本视频演示了一种符合这些规范的检测方法, 即使用尿液或血清 (为病人) 或被粉碎的蚊子尸体 (用于环境监测), 所有这些都没有大量的样品准备。在运载季铵盐组 (Q 纸) 的纸上捕获蚊子的尸体, 然后用氨水处理来管理生物危害。这些都是直接的, 没有 RNA 分离, 放入化验管中含有冷冻干燥试剂, 无需冷藏链。改进形式的反向转录回路介导的等温放大与目标特定的荧光标记不可替代探针产生读数, 在30分钟, 作为一个三色荧光信号。这是可视化的手持式电池供电设备与橙色过滤器。前污染是防止与密封管, 并使用 thermolabile 尿 DNA glycosylase (UDG) 在存在 dUTP 在放大混合物。

首先, 用凝胶电泳法对各 RT 灯底漆(表 1)及其相应的病毒 RNA 基底和阴性对照进行了性能评估。用于 Zika 和登革1的 NS5 区域 (rna 依赖性 rna 聚合酶) 和 nsP2 区域 (非结构化蛋白 P2) 用于基孔肯亚的 RT 灯引物。模板是从非洲绿猴肾脏 (维罗) 细胞培养的病毒种群中提取的总 RNA。在一种情况下, 从基孔肯亚感染的雌性Ae 蚊 (表 2)中提取总核酸 (DNA 和 RN...

Watch this Scientific Journal Video about Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1 at JoVE.com

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

目前用于检测 Zika、基孔肯亚和登革热病毒的复用诊断程序需要复杂的样本准备和昂贵的仪器, 而且在低资源环境下很难使用。我们显示一个诊断, 使用等温放大与目标特定的链不可替代探针, 以检测和区分这些病毒具有高灵敏度和特异性。

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

Research

JoVE Journal

Biochemistry

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

制造的UV-Vis和拉曼光谱免疫平台

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, ...

科研

生物化学

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

电化学检测氘动力学同位素对希瓦氏菌 oneidensis MR-1 细胞外电子传输的影响

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

体外检测 tRNA-Isopentenyl 转移酶活性的实验研究

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

大鼠甲基 Seq 平台识别与应力接触相关的表观变化

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

基于原生自荧光的sFLIM测量植物部分荧光探针和木质素之间的相互作用

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

使用多路复用共免疫沉淀对蛋白质相互作用网络动力学进行量化

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

一个集成拉曼光谱和质谱平台，用于研究单细胞药物接受、代谢和效果

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in ...

Platform Incubator with Movable XY Stage: A New Platform for Implementing In-Cell Fast Photochemical Oxidation of Proteins

具有可移动XY阶段的平台孵化器：实现蛋白质细胞内快速光化学氧化的新平台

Fast Photochemical Oxidation of proteins (FPOP) coupled with mass spectrometry (MS) has become an invaluable tool in structural proteomics to interrogate ...