Name: visual detection of multiple nucleic acids in a capillary array
Uploaded: 2017-11-15T14:00:00
Description: Watch this Scientific Journal Video about Visual Detection of Multiple Nucleic Acids in a Capillary Array at JoVE.com

本研究部分由国家自然科学基金资助 (31370813、3147670、31670831和 31600672)、国家转基因植物专项基金 (2016ZX08012-003、2016ZX08012-005)、新世纪优秀人才项目大学, 中国国家重点研究开发项目 (2016YFA0500601) 和中国博士后科学基金 (2016M591667)。

注意: 本协议假定已为所需的微通道和加载适配器提供形状的不锈钢模具 (3D 文件为 补充文件 1 和 2).该协议还假定已经进行了植物 DNA 分离. 
 1. 制作毛细管阵列现成的盒式磁带 <ol> <li> 清洗不锈钢模具。
	<ol> <li> 通过洗涤剂、乙醇和去离子水清洗不锈钢模具, 去除潜在的污染物。用氮气烘干模具. </li>
	</ol> </li> <li> 清除毛细管 ...

<ol>
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这里展示的平静平台, 它结合了灯技术与毛细管阵列, 使同时检测多个转基因相关的基因目标在一个单一的, 高效, 易于操作的测试。
为了在卡带中成功地执行复用灯的反应, 需要注意三关键点。首先, 达到相同的高度, 在毛细血管的上部和亲水性和疏水性模式的毛细管阵列是关键的同时加载试剂到所有的毛细血管。在初始插入到 "支持" 后, 毛细血管应由一个板对齐, 以确保它们?...

在广泛的领域, 如临床诊断1、2、3、转基因检测4, 对多种核酸同时检测的系统来说, 需要低成本、快速且易于使用. 5,6, 微生物监视7,8,9, 鉴证分析10,11, 特别是点测试 (POCTs), 其中资源通常被限制为12,13,14。
聚合酶链反应 (pcr), 包括其衍生物方法 real-time pcr 和多重 pcr, 是最广泛应用于这些领域的检测技术。但是, 这些方法通常只在一个测试15中检测一个目标, 并且需要电力和复杂的专业设备。
另一个有前途的技术检测核酸是环介导的等温放大 (灯), 这是第一次描述在 2000年16。灯是一种高效的 DNA 检测方法。理论上, 它可以放大1拷贝到 109副本的增在一个小时内, 所有执行在一个恒定的温度, (即, 在 60-65 ° c)。成功的放大将产生大量的不溶性副产品焦磷酸盐和导致混浊的变化17, 这可以直接观察肉眼。还可以通过添加金属离子或荧光染料 (如素18、核酸染料19和羟基萘酚蓝20) 来观察颜色的变化。由于光源具有灵敏度高、操作方便等优点, 在核酸检测中得到了广泛的应用。
目前, 多路灯检测主要有两种策略。一是通过在一管21,22,23中的多个灯引套进行多灯检测。然而, 由于不同引物间的内在干扰和竞争, 其多样性和放大效率将受到限制。此外, 在同一反应中, 很难识别出不同的灯具产品。另一个策略是基于物理隔离。不同的引物集合被隔绝了入各自的小型化隔间, 并且多个灯反应然后同时执行24,25。这些方法, 通常是基于微流控芯片, 提供了一个潜在的解决方案的高通量灯的反应。然而, 生产的芯片和复合预的底漆集是复杂的, 这可能会增加成本和减少重复性。
最近, 有几项研究描述了在毛细血管中执行灯反应以绕过复杂的微流控芯片制造, 并实现了低成本检测26,27。然而, 在高通量分析中, 这些毛细血管与 PCR 条管的微型版本类似, 因为样品和反应试剂 (包括不同的底漆) 必须单独准备并送到不同毛细血管内的反应单位。为了实现并行和复用分析, 需要额外的设备, 例如多通道注射器泵, 是平行装载样品或试剂。
为了克服目前核酸多重检测方法的局限性, 我们开发了一种将视灯技术与毛细管阵列相结合的小型化平台。该平台具有多目标、体积小巧、成本低廉、易于操作的28。在此, 我们描述了如何制造毛细管阵列和执行在阵列中的灯反应的细节。这里描述的协议已被标准化使用转基因生物 (转基因) 检测作为一个模型。重要的是, 这个协议也可以用于高通量检测其他核酸靶。

<table>
	<tbody>
		<tr>
			<td>UltraEverDry(super-hydrophobic coat)</td>
			<td>UltraTech</td>
			<td>4001</td>
			<td>supplier:Exiron chemistry(CHINA) CO.,LTD.</td>
		</tr>
		<tr>
			<td>PDMS</td>
			<td>Dow Corning</td>
			<td>8332557</td>
			<td></td>
		</tr>
		<tr>
			<td>Bst polymerase</td>
			<td>New England BioLabs</td>
			<td>M0275L</td>
			<td></td>
		</tr>
		<tr>
			<td>betain</td>
			<td>Sigma-Aldrich</td>
			<td>B0300-1VL</td>
			<td></td>
		</tr>
		<tr>
			<td>calcein</td>
			<td>Sigma-Aldrich</td>
			<td>C0875-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>MnCl2</td>
			<td>Sigma-Aldrich</td>
			<td>MKBP0495V</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>New England BioLabs</td>
			<td>B1003S</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>Shanghai Sangon</td>
			<td>B804BA0022</td>
			<td></td>
		</tr>
		<tr>
			<td>chitosan</td>
			<td>Shanghai Sangon</td>
			<td>LJ0805S309J</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoshop 7.0 software</td>
			<td>Adobe Systems Inc., CA, USA</td>
			<td></td>
			<td>Image analysis</td>
		</tr>
		<tr>
			<td>GenePix Pro 6.1</td>
			<td>Molecular Devices, CA, USA</td>
			<td></td>
			<td>microarray analysis software</td>
		</tr>
		<tr>
			<td>AutoCAD</td>
			<td>Adobe Systems Inc.</td>
			<td></td>
			<td>3D construction software</td>
		</tr>
		<tr>
			<td>UV filter (ZWB2)</td>
			<td>YXSensing</td>
			<td></td>
			<td>supplier : taobao</td>
		</tr>
	</tbody>
</table>

visual detection of multiple nucleic acids in a capillary array

在疾病诊断、微生物监测、转基因生物体 (转基因) 检测中迫切需要多目标、短期和资源可承受的多种核酸检测方法, 并法医分析我们先前描述的平台称为平静 (Capillary Array-based Loop 介导的等温放大为Multiplex 视觉检测核酸)。在此, 我们描述了改进的制造和性能的过程, 这个平台。在这里, 我们应用一个小的, 现成的磁带组装的毛细管阵列的多重视觉检测核酸。毛细管阵列被预处理成疏水和亲水模式之前固定环介导的等温放大 (灯) 底漆集在毛细管。加载适配器组装后, 灯反应混合物被加载和孤立到每一个毛细管, 由于毛细管力由一个单一的移步骤。灯的反应在毛细血管中平行进行。结果是视觉读出的照明与 hand-held 紫外线手电筒。利用这个平台, 我们展示了对8种常见的元素和基因在转基因样品中具有高特异性和敏感性的监测。总之, 本文所描述的平台是为了方便检测多种核酸。我们相信它将广泛应用于需要高通量核酸分析的领域。

在该方法中, 在试样加载过程中防止不同毛细管间的交叉污染是非常重要的。为此, 引入了壳聚糖, 可以保留单个毛细血管中的底漆。为了测试它是否工作, 我们 pre-fixed ADH1 (玉米内源性参考基因) 在毛细管盒中设置的 "T" 和 "U" 的模式, 如 图 3a中所示。如预期的那样, 只有毛细血管中包含了显示阳性信号 ( 图 3b</...

Watch this Scientific Journal Video about Visual Detection of Multiple Nucleic Acids in a Capillary Array at JoVE.com

Visual Detection of Multiple Nucleic Acids in a Capillary Array

毛细管阵列中多核酸的视觉检测

该协议描述了一个小的, 现成的卡带的制作, 可用于视觉检测的多个核酸在一个单一的测试, 这是易于操作。在这种方法中, 用毛细管阵列对转基因生物的目标进行多重和高效的检测。

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Research

JoVE Journal

Bioengineering

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

科研

生物工程

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

对芯片等速电泳离子的分离和纯化核酸

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

A Microfluidic Device for Studying Multiple Distinct Strains

一种微流体装置研究多个不同的菌株

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Capillary Force Lithography for Cardiac Tissue Engineering

毛细力光刻技术在心脏组织工程

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Polymeric Microneedle Array Fabrication by Photolithography

聚合物微针阵列制造用光刻

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

微孔阵列平台中微生物群落发展的组装与跟踪

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

多路人工细胞微环境阵列的制备

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

使用一个荧光显微镜进行多种成像模式

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

多平行板流室对剪切应力暴露的内皮细胞基因表达分析

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...