Name: reverse transcription-loop-mediated isothermal amplification (rt-lamp) assay for zika virus and housekeeping genes in urine, serum, and mosquito samples
Uploaded: 2018-09-14T15:00:00
Description: Watch this Scientific Journal Video about Reverse Transcription-Loop-mediated Isothermal Amplification RT-LAMP Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples at JoVE

这项工作得到了莫林和罗纳德-神经家庭慈善捐款和密歇根州圣玛丽的实地研究所的支持。资助者在研究设计、数据收集和分析、决定出版或编写手稿方面没有任何作用。我们还感谢伯纳黛特 Zwaans 博士和以利亚沃德对手稿的批判性审查。

这里描述的所有方法都已得到博蒙特健康机构审查委员会 (IRB) 的批准。所有的实验都是按照相关的指导方针和规定进行的。
注意: 所有可能感染的材料应按照生物安全等级2标准处理, 包括使用个人防护设备。任何可能产生气溶胶的程序都应在生物安全柜中进行。此外, 应在适当的节肢动物控制1-3 级设施中进行活蚊的工作。鉴于 ZIKV 感染与先天性畸形的关系, 怀孕、试图受孕的...

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本文所描述的 ZIKV RT 灯的研究工作使用的人和蚊子样本6。检测的限制约为1基因组等效6, 这应该是足够的, 因为典型的病毒负荷的症状 ZIKV 感染患者是 10 3 至 106 PFU/毫升7。此外, 该方法可以检测不先分离 RNA 的样品中的 ZIKV, 细胞培养中不存在病毒放大。与 qRT PCR 相比, 这大大降低了这种检测的时间、成本和健康风险。</p...

在 2015年, Zika 病毒 (ZIKV) 作为一种传染性疾病引起了全球瞩目, 因为怀孕期间的感染与流产、死胎、严重的神经系统先天缺陷 (包括小头) 以及其他先天性出生缺陷1。在极少数情况下, ZIKV 与格林-巴利综合征有关。ZIKV 主要由白纹蚊传播;然而, 它也可以通过性接触传播1。鉴于感染 ZIKV 的人在多数人中无症状, 或出现轻度流感样症状, 与其他 arborviruses1感染的症状重叠, 因此需要改进方法, 快速和经济高效地检测ZIKV, 以筛查人和当地蚊子的数量。
定量反向转录 pcr (qRT pcr) 是一种可靠的 ZIKV 检测方法;然而, 这种技术需要昂贵的专业设备, 训练有素的人员, 和 RNA 分离从样本的兴趣。反向转录回路介导的等温放大 (RT 灯) 是一种基于 PCR 技术的一步核酸扩增方法, 它只需要一个孵化温度由于使用了嗜热 DNA 聚合酶与链位移特性。这就绕过了对 thermocycler 的需要, 减少了完成化验所需的时间长短。RT 灯的其他优点包括其高度特异性和灵敏度, 在 pH 水平和温度范围内的鲁棒性, 以及对许多 PCR 抑制剂2的抗性。它的成本相对较低, 试剂在室温下稳定。鉴于这些特点, RT 灯可以部署在实验室或现场。因此, 已经开发了灯反应, 以检测一系列的病原体和其他类型的感染3,4,5。本文所描述的 RT 灯协议的目标是检测在人体血清和尿样中没有 RNA 分离的 ZIKV, 以及通过 RT 灯在30分钟以内的单一感染蚊子。该方法可用于替代 qRT PCR, 因为它是一种灵敏的, 快速的诊断工具, 工作迅速和在实验室以外的设置。

<table border="0" cellpadding="0" cellspacing="0" style="width:901px;" width="902">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Bst 2.0 WarmStart DNA polymerase</td>
			<td style="width:223px;">New England Biolabs</td>
			<td style="width:223px;">M0537S</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isothermal Amplification Buffer&#160;</td>
			<td>New England Biolabs</td>
			<td style="width:223px;">B0537S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">WarmStart RTx Reverse Transcriptase</td>
			<td>New England Biolabs</td>
			<td style="width:223px;">M0380S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antarctic Thermolabile UDG</td>
			<td>New England Biolabs</td>
			<td style="width:223px;">M0372S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4</td>
			<td>New England Biolabs</td>
			<td style="width:223px;">B1003S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100mM dATP Solution</td>
			<td>New England Biolabs</td>
			<td style="width:223px;">N0440S</td>
			<td>Deoxynucleotide Set N0446S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100mM dCTP Solution</td>
			<td>New England Biolabs</td>
			<td style="width:223px;">N0441S</td>
			<td>Deoxynucleotide Set N0446S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100mM dTTP Solution</td>
			<td>New England Biolabs</td>
			<td style="width:223px;">N0443S</td>
			<td>Deoxynucleotide Set N0446S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100mM dGTP Solution</td>
			<td>New England Biolabs</td>
			<td style="width:223px;">N0442S</td>
			<td>Deoxynucleotide Set N0446S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100mM dUTP Solution</td>
			<td style="width:223px;">Thermo Fisher Scientific</td>
			<td style="width:223px;">R0133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYBR Green I Nucleic Acid Gel Stain</td>
			<td style="width:223px;">Invitrogen</td>
			<td style="width:223px;">S7563</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nancy-520</td>
			<td style="width:223px;">Sigma Aldrich</td>
			<td style="width:223px;">01494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low DNA Mass Ladder</td>
			<td style="width:223px;">Invitrogen</td>
			<td style="width:223px;">10-068-013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Zika Postive Control</td>
			<td style="width:223px;">Robert Koch Institute, Germany</td>
			<td style="width:223px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Agarose</td>
			<td style="width:223px;">Thermo Fisher Scientific</td>
			<td style="width:223px;">BP1600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">BlueJuice Gel Loading Buffer</td>
			<td style="width:223px;">Invitrogen</td>
			<td style="width:223px;">10816015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Primers</td>
			<td style="width:223px;">Integrated DNA Technologies</td>
			<td style="width:223px;">Custom Oligo</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Nuclease-Free Water</td>
			<td style="width:223px;">Ambion</td>
			<td style="width:223px;">AM9938</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Urine Preservative</td>
			<td style="width:223px;">Norgen Biotek</td>
			<td style="width:223px;">18126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">ZIKV PCR Standard</td>
			<td>Robert Koch Institute</td>
			<td style="width:223px;">N/A</td>
			<td>Vero E6 cell supernatants</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">DENV2 New Guinea C Control</td>
			<td style="width:223px;">Connecticut Agricultural Experiment Station</td>
			<td style="width:223px;">N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

reverse transcription-loop-mediated isothermal amplification (rt-lamp) assay for zika virus and housekeeping genes in urine, serum, and mosquito samples

感染 Zika 病毒 (ZIKV) 可能是无症状的成人, 但是, 在怀孕期间感染可能导致流产和严重的神经系统出生缺陷。该协议的目标是快速检测人类和蚊子样本中的 ZIKV。目前 ZIKV 检测的金标准是定量反向转录 pcr (qRT pcr);反向转录回路介导的等温放大 (RT 灯) 可能允许更有效和低成本的测试, 而不需要昂贵的设备。在本研究中, RT 灯用于在30分钟内在各种生物样品中 ZIKV 检测, 而不首先从样品中分离出 RNA。这种技术是使用 ZIKV 感染的病人尿液和血清, 以及感染的蚊子样本。18S. 核糖体核糖核酸和肌动蛋白分别用作人体和蚊子标本的对照。

可以用三种不同的方法分析 RT 灯的反应。首先, 随着荧光核酸染料的加入, 阳性反应将呈黄色/绿色, 在这种颜色中, 阴性反应会呈橙色到肉眼的颜色。第二, 将荧光核酸染料添加到 RT 灯反应中, 当样品被紫外光激发时, 会产生荧光信号。阴性反应将不会有一个可检测的荧光信号在任何背景荧光, 可能存在的小剂量在负控制。最后, 在琼脂糖凝胶上可以用到 RT 灯的反应。阳性的...

Watch this Scientific Journal Video about Reverse Transcription-Loop-mediated Isothermal Amplification RT-LAMP Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples at JoVE

Reverse Transcription-Loop-mediated Isothermal Amplification RT-LAMP Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

反向转录-环介导的等温放大 (RT 灯) 测定尿液、血清和蚊子标本中的 Zika 病毒和管家基因

该协议提供了一种高效、低成本的方法, 用于检测人体尿液、血清样品或蚊子中的 Zika 病毒或控制靶, 采用反向转录环介导的等温放大 (RT 灯)。此方法不需要 RNA 隔离, 可以在30分钟内完成。

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth ...

此方法可以快速确定样品是否含有寨卡病毒。这对于孕妇、试图怀孕的夫妇或监测有寨卡感染风险的当地蚊子种群非常有用。该技术的主要优点是，它快速、简单，不需要昂贵的设备，并且无需RNA隔离步骤即可完成。该技术的影响延伸到寨卡感染的诊断，因为怀孕期间的感染与流产、死产和其他严重的先天性神经性先天缺陷有关。虽然这种方法可以提供对寨卡的洞察，但它也可以通过使用病毒特异性底法应用于其他传染病，如登革热、基孔肯雅病毒或西尼罗河病毒。首先，将分子级水中的每个RT-LAMP底图重新构成100微摩尔的最终浓度。简要涡流底解，以确保溶液是均匀的。以最高速度离心约五秒钟，以收集底转溶液。然后准备一个 10x RT-LAMP 底像组合，如技术协议表二中所述。短暂涡流，确保均匀性。并在最大速度下短暂离心，以避免任何损失。将底像存放在零下20摄氏度的高温下，避免任何冻结循环。对于人体尿液样本，使用新鲜尿液、冷冻尿液或防腐剂。使用前在冰上解冻任何冷冻样品。人类血清样本应新鲜或冷冻，而ZIKV感染的细胞系应含有条件介质或细胞酸盐。最后，对于伊蚊，使用整个尸体，无论是新鲜或冷冻。在冰上解冻任何冷冻蚊子尸体。将单个蚊子放在 100 微升 PBS 中。使用P10移液器尖端粉碎蚊子10次，并均匀化它，以产生一个粗糙的蚊子的lysate。简要地将粗液分离成颗粒任何碎屑，并使用上流液进行下游RT-LAMP分析。首先，为要使用的每个底像准备的RT-LAMP主混合反应，如技术协议中的表三所示。漩涡每个样品，以确保它很好地混合。然后简要旋转它们，以防止体积丢失。对于每次反应，RT-LAMP 主液的移液器 23.0 微升混合成 200 微升 PCR 管。然后，添加两个微升样品，使总体积达到 25.0 微升。使用分子级水进行负对，并确保包括阳性对照。可选地，包括相关阿尔博病毒（如登革热病毒）的特异性控制反应，如技术协议中所述。使用热块、水浴或热循环器，在 61 摄氏度下加热样品 30 分钟。在此之后，将热量增加至80摄氏度10分钟，以加热停用聚合酶。在单独的封闭空间中执行 RT-LAMP 分析。在 TAE 缓冲液中稀释荧光核酸染料一至十。在RT-LAMP反应的12个微升器中加入荧光核酸染料溶液的两个微升器。通过查找颜色变化的存在，直观地评估 RT-LAMP 反应。可选使用相机在白色背景上拍摄 RT-LAMP 反应的照片。接下来，将样品放在302纳米的紫外光下，通过荧光确认RT-LAMP产品的存在。使用相机拍摄结果。开始执行电泳将 2% 的 agarose 凝胶倒入 1x TAE 缓冲液中，并含有核酸污渍，用于可视化。在第一条通道中加入五个微升的DNA梯子，比较分子量。对于每个RT-LAMP反应，将13微升RT-LAMP反应混合物与两个微升DNA加载染料混合。加载这 15 微升混合物。以 90 伏运行凝胶 90 分钟，或直到带子分离。具有 302 纳米紫外光的图像。完成后，将RT-LAMP反应处理在双密封袋中。本研究采用三种不同的方法分析RT-LAMP反应。使用荧光核酸染料时，阳性反应呈黄绿色，而负反应呈橙色。当样品被紫外线点燃时，这种染料也会产生荧光信号。最后，RT-LAMP反应可以在阿加罗斯凝胶上耗尽。正RT-LAMP反应将有一个带状模式，而负反应将没有DNA带。这些方法用于演示ZIKVRT-LAMP反应的特异性。只有ZIKV分子控制具有阳性的RT-LAMP反应。此外，虽然ZIKV在已知ZIKV感染患者的尿液和血清中均被检测到，但在没有ZIKV感染的无症状控制患者中却未发现ZIKV。然后对样品进行人体18S rRNA测试。只有从患者身上采集的样本对18S rRNA呈阳性反应。蚊子样本也可以对 ZIKV 或 RT-LAMP 质量控制 AEDAE 进行类似测试。只有ZIKV和感染ZIKV的蚊子的阳性分子对照对ZIKV呈阳性。然而， 所有的蚊子都积极为 Aedae 由 Rt - lamp 。一旦掌握，这项技术可以在30分钟内完成，如果它执行得当。重要的是要记住，RT-LAMP反应容易出现误报反应。预防步骤包括在反应中使用热实验室DNA糖基酶，使用横向工作流程，在单独的空间中执行分析，以及减少扩增管打开后的时间。按照此过程，可以执行其他方法（如 QPCR）以回答其他问题，例如示例中存在多少个副本的病毒或其他目标。看完这段视频后，您应该对如何使用RT-LAMP检测寨卡病毒和人体尿液和血清以及蚊子中的目标控制有一个很好的理解。不要忘记，使用寨卡病毒和核酸污渍可能极其危险，在执行此过程时应始终采取戴手套等预防措施。鉴于寨卡病毒感染与先天性异常有关，怀孕、试图怀孕的妇女或这些妇女的伴侣应显著减少她们在实验室中感染寨卡病毒的风险。

Research

JoVE Journal

Genetics

Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

诱导体细胞行业分析（ISSA）的为研究木材形成和二次开发茎相关基因和推动者使用

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little ...

科研

遗传学

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

肽衍生的方法在运输跨越植物细胞的基因和蛋白质和细胞器障碍

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

用新的转介导法测定大肠杆菌中 DNA 元素的最佳染色体位置 (s)

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

低丰度胚胎标本的染色质沉淀 (芯片) 协议

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

昆虫细胞 (sf9) 外源基因在蛋白质功能检测中的瞬态表达

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer

3 ' 快速扩增 CDNA 末端, 以图记录癌症

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the ...

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

用于下一代测序的近全长 HIV-1 Proviruses 的扩增

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

一种通用嵌套逆转录酶链反应检测裂解病毒的评价

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

用于评估肿瘤细胞中的微RNA水平、功能和相关靶基因的体外协议

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...