Name: visual detection of multiple nucleic acids in a capillary array
Uploaded: 2017-11-15T14:00:00.000+00:00
Duration: 8 min 56 s
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> 戴上安全护目镜、实验室和 #160; 制服和化学防护手套. <li>
		<li> 将毛细管放入烧杯中。用30毫升丙酮清洁毛细血管5分钟去除有机物, 然后用去离子水冲洗毛细血管. 
		注意: 在油烟罩中处理丙酮. </li>
		<li> 将10毫升 h 2 O 2 放入烧杯和 #160; 然后将 30 ml h 2 , 所以 4 加入到 h 2 O 2 中, 以防止过热。确保解决方案 (食人鱼溶液) 完全覆盖毛细血管至少30分钟. 
		注意: 在处理食人鱼溶液时要小心 (H 2 , 以便 4 /h 2 O 2 = 3: 1, v/五) 。如果食人鱼溶液溢出, 用大量的水迅速冲洗溶液, 并用纸巾擦拭表面. 
		注: 对毛细管进行彻底清洗是保证灯反应成功的关键之一。在清洗过程中必须摇动酸缸, 去除毛细管中的气泡, 必要时也可延长清洗时间. </li>
		<li> 用大量的水冲走食人鱼溶液。用乙醇和去离子水冲洗毛细血管5分钟。在干燥的烤箱中干燥毛细管. </li>
	</ol> </li> <li> 倒烷 (硅橡胶) <ol> 重出了在50毫升管中有1:1 的比值的硅橡胶底座和硫化试剂。通常, 混合5克的弹性体基地, 以5克的弹性体固化剂. </li>
		<li> 用玻璃棒将混合物彻底搅拌约5分钟。将该管与硅橡胶在真空钟罐中放置30分钟吸管. </li>
		<li> 将其慢慢倒入不锈钢模具的气缸中。允许硅烷在60和 #176 的干燥炉中固化3小时; C 注意: 在浇注时要小心避免气泡。在浇注后, 让它站在5分钟的自然去除的气泡出的硅橡胶. </li>
	</ol> </li> <li> 在硫化硅橡胶后将模具从 "硅橡胶" <ol> 中卸下后, 通过用气缸拉出模具来拆卸模具。用手术刀把模具的边缘切掉, 从气缸中取出. </li>
		<li> 用乙醇和去离子水清洗三次。用氮气烘干的支持. </li>
	</ol> </li> <li> 将该支持的下表面视为疏水性。在室温下, 用疏的外套浸泡1秒的硅橡胶支架. </li> 干燥10分钟
	<li> 将毛细管插入到 "支持" <ol> <li> 中, 将已清洗的4毫米毛细管插入到该支持的孔中, 并将0.5 毫米的毛细管留在该支持的顶面外。确保毛细血管的两端处于同一水平. </li>
	</ol> </li> <li> 将毛细管的外表面和该支持的顶面表面视为疏水性. 
	<ol> <li> 添加15和 #181; 我疏大衣上表面的支持。请注意, 涂层立即蔓延到所有的顶部表面 (包括表面的支持和外部表面的毛细血管外露部分) 的毛细管力. </li>
		<li> 空气干燥疏改性毛细管阵列. </li>
	</ol> </li> <li> 将底漆固定到毛细管阵列 <ol> 准备底漆溶液中。重出0.65 克壳聚糖 (分子式: (C 6 H 11 无 4 ) n ), 并将其分解为50毫升去离子水, 通过调节 pH 值为 4.5-5.5, 用乙酸实现壳聚糖浓度为1.3%。根据 表 1 准备底漆组件组合。请参见 辅助表 1 以引物 </li> <li> 添加1.6 和 #181; 一组底漆的混合物, 根据预先设计好的顺序填充毛细管阵列的一个相应毛细管. 
		注: 其余的两个空白毛细管被设置为负控制. </li>
		<li> 将阵列锚定在标准平底96井板的透明井中, 并在60和 #176 处干燥阵列; C 至少 2 h. </li>
	</ol> </li> </ol> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"> <tbody> <tr> <td> 灯具底漆固定混合组件 (初始浓度) </td> <td> 音量 (#956; L) </td> </tr> <tr> <td> ddH 2 O </td> <td> 17.0 </td> </tr> <tr> <td> 壳聚糖 (1.3%) </td> <td> 1.0 </td> </tr> <tr> FIP/毕普底漆 (20 和 #956; m) </td> <td> 2.0/2.0 </td> </tr> <tr> <td> LoopF/LoopB 底漆 (20 和 #956; m) </td> td>> 1.0/1.0 </td> </tr> <tr> <td> F3/B3 入门 (20 和 #956; M) </td> <td> 0.5/0.5 </td> </tr> <tr> <td> 总卷 </td> <td> 25.0 </td> </tr> </tbody> </table> 表 1: 灯的组成部分底漆固定试剂. 在表的左列中列出了灯具底漆固定组合件的组成, 并在右列中列出了每个组件的音量. 
<ol start="9"> <li> 按如下方式组装卡带。将加载适配器放到固定阵列的顶部。确保适配器的碟只覆盖所有十毛细血管暴露的部分 (参见 图 1 ). </li>
</ol> <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56597/56597fig1.jpg"/> 图 1: 磁带制作和组装。( a ) 不锈钢模具和硅橡胶支持。模具由3部分组成: 汽缸、坝板、柱板。( b ) 支持制造和组装毛细管盒的电路原理图。整个过程包含5步骤: 1. 在浇注, 2. 脱模, 3. 毛细管插入和表面涂层, 4. 底漆固定和5。磁带锚定。1. 将硅橡胶倒入模具的气缸中;2. 推出大坝板, 从支持的模式中取出模具;3. 将支持的下表面涂上涂层, 然后将毛细管插入到该技术支持中, 最后涂上表面的支持和暴露的毛细血管。粗蓝线表示疏外套;4. 将底漆装入单独的毛细血管;5. 将毛细管阵列固定在单96孔板上, 并在其上安装一个样品加载适配器。详细信息已在协议步骤 1.1-1.9 中描述。<a href="//ecsource.jove.com/files/ftp_upload/56597/56597fig1large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a> 
 2. 在毛细管阵列中灯反应的性能 <ol> <li> 准备反应组合 <ol> <li> 准备灯的反应混合, 每 T 能 2 . </li>
		<li> 添加试剂到混合物根据所选择的顺序, 如在 表 2 , 和涡流的反应混合物 5 s 后添加素。轻轻地在在添加 Bst 聚合酶后20次垂直于试管. </li>
	</ol> </li> </ol> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"> <tbody> <tr> <td> 灯组件和 #160; #160; #160; #160; #160; #160; #160; #160; #160; #160; #160; #160; #160;和 #160; (初始浓度) </td> <td> 卷 (和 #956; L) </td> </tr> <tr> <td> ddH MgSO </td> <td> </td> 11.6 </tr> <tr> <td> , 4 (100 mM) </td> <td> 2.0 </td> </tr> <tr> <td> dNTPs (25 mm) </td> <td> 1。4d > </tr> <tr> <td> 甜菜碱 (5 米) </td> <td> 4.0 </td> </tr> <tr> <td> 缓冲 (10x) </td> <td> 2.5 </td> </tr> <tr> <td> 素 (1.25 mM) </td> <td> 0.5 </td> </tr> <tr> <td> MnCl 2 (25 mM) </td> <td> 0.5 </td> </tr> <tr> <td>/em > 聚合酶 (8 U/和 #956; l) </td> <td> 1.5 </td> </tr> <tr> <td> 植物 DNA (10 ng/和 #956; l) </td> <td> 1.0 </td> </tr> <tr> 总卷 <td> </td> 25.0 <td> </td> </tr> </tbody> </table>表 2: 毛细管阵列灯的反应系统。 在左列中列出了毛细管阵列灯组件的反应系统的组件, 并在右列中列出了每个组件的音量. 
<ol start="2"> <li> 加载试剂并密封卡带 <ol> <li> 吸管20和 #181; 与标准的100和 #181 的灯反应混合物; 我的小费。将笔尖插入加载适配器的入口以锁定它。将反应混合物轻轻地注入适配器的碟中;反应混合物会迅速填满盘子, 然后通过毛细管力自动加载毛细管. </li>
		<li> 卸下带有锁定尖端的适配器, 并通过与 PCR 兼容的透明密封膜密封该井. 
		注: 只有在亲水性盘中接触反应混合物的毛细血管才能填充。因此, 请确保所有毛细管的顶部都具有相同的高度 (请参见 图 2 ). </li>
	</ol> </li> </ol> <img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56597/56597fig2.jpg"/> 图 2: 使用加载适配器进行采样加载的图示. 图片显示使用蓝色解决方案的加载过程为例。将笔尖插入进风口, 然后缓慢地将样品注入适配器, 然后用锁定的笔尖卸下适配器。<a href="//ecsource.jove.com/files/ftp_upload/56597/56597fig2large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a> 
<ol start="3"> <li> 在63和 #176 的恒温箱中孵育毛细管阵列; C 用于1小时 </li> </ol>
 3. 结果读出和数据分析 <ol> <li> 获取荧光发射的图像. 
	<ol> <li> 在小型 hand-held uv LED 手电筒的顶面上修复 uv 滤镜, 以过滤可见光. </li>
		<li> 激发离解的素发出荧光与紫外线手电筒。通过数码相机或智能手机从毛细管阵列顶端捕获图像. </li>
		<li> 拍摄图像时, 确保摄像机在毛细血管的区域内尽可能地放大, 以获得高质量、清晰的图像. </li>
	</ol> </li> <li> 分析结果 <ol> <li> 通过图像分析软件打开图像, 然后选择和 #34; 图像和 #62; 模具和 #62; 灰度和 #62; 是和 #34; 和 #34; 图像和 #62; 模具和 #62; 16bit 和 #62; 是和 #34;选择和 #34; 文件和 #62; 保存 #62; 格式和 #62; TIFF 和 #34; 将图像转换为16位 tiff 格式. </li>
		<li> 提取荧光强度值 <ol> <li> 打开微阵列分析软件。将16位 TIFF 格式图像拖动到软件的界面中, 然后选择波长 #34; 532 nm 和 #34; 以及 #34、绿色和 #34 的颜色; 显示图像. </li>
			<li> 创建一个新的块来定位来自毛细血管的荧光信号。选择和 #34; 工具和 #62; 新的块和 #34; 并输入列和行数, #34; 1; 1 和 #34; 和 #34; 2; 5 和 #34; 在和 #39; 块和 #39; 和 #39; 功能和 #39; 接口分开. </li>
			<li> 右键单击并选择和 #34; 功能和 #34; 模型, 然后调整位置和直径, 以适应毛细血管的荧光区域。选择和 #34; 分析和 #34; 提取荧光强度的值. </li>
			<li> 选择和 #34; 文件和 #62; 保存设置为和 #34; 保存块文档, 选择和 #34; 文件和 #62; 保存结果和 #34; 保存荧光强度结果。计算信噪比, 以确定是否成功地在毛细管中执行灯. 
			注: 通过记录斑点的灰度值和信噪比 (棚), 得到了毛细管的信号, 定义为目标的前景信号的平均值与两个负的前向信号平均值的比值。控件.确定正信号的截止阈设置为信噪比和 #62; 2. </li>
		</ol>
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D., Kessel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+Ebola+tells+us+about+outbreak+diagnostic+readiness.">What Ebola tells us about outbreak diagnostic readiness.</a> Nat Biotechnol. 33 (5), 464-469 (2015).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+universal+multiplex+PCR+strategy+for+100-plex+amplification+using+a+hydrophobically+patterned+microarray.">A universal multiplex PCR strategy for 100-plex amplification using a hydrophobically patterned microarray.</a> Lab Chip. 11 (21), 3609-3618 (2011).</li><li>Notomi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+of+DNA.">Loop-mediated isothermal amplification of DNA.</a> Nucleic Acids Res. 28 (12), (2000).</li><li>Mori, Y., Nagamine, K., Tomita, N., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+loop-mediated+isothermal+amplification+reaction+by+turbidity+derived+from+magnesium+pyrophosphate+formation.">Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation.</a> Biochem Biophys Res Commun. 289 (1), 150-154 (2001).</li><li>Tomita, N., Mori, Y., Kanda, H., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+(LAMP)+of+gene+sequences+and+simple+visual+detection+of+products.">Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products.</a> Nat Protoc. 3 (5), 877-882 (2008).</li><li>Iwamoto, T., Sonobe, T., Hayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+for+direct+detection+of+Mycobacterium+tuberculosis+complex,+M-avium,+and+M-intracellulare+in+sputum+samples.">Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M-avium, and M-intracellulare in sputum samples.</a> J Clin Microbiol. 41 (6), 2616-2622 (2003).</li><li>Goto, M., Honda, E., Ogura, A., Nomoto, A., Hanaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+detection+of+loop-mediated+isothermal+amplification+reaction+by+using+hydroxy+naphthol+blue.">Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue.</a> Biotechniques. 46 (3), 167-172 (2009).</li><li>Iseki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+multiplex+loop-mediated+isothermal+amplification+(mLAMP)+method+for+the+simultaneous+detection+of+bovine+Babesia+parasites.">Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites.</a> J Microbiol Methods. 71 (3), 281-287 (2007).</li><li>Liang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+loop-mediated+isothermal+amplification+detection+by+sequence-based+barcodes+coupled+with+nicking+endonuclease-mediated+pyrosequencing.">Multiplex loop-mediated isothermal amplification detection by sequence-based barcodes coupled with nicking endonuclease-mediated pyrosequencing.</a> Anal Chem. 84 (8), 3758-3763 (2012).</li><li>Shao, Y., Zhu, S., Jin, C., Chen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+multiplex+loop-mediated+isothermal+amplification-RFLP+(mLAMP-RFLP)+to+detect+Salmonella+spp.+and+Shigella+spp.+in+milk.">Development of multiplex loop-mediated isothermal amplification-RFLP (mLAMP-RFLP) to detect Salmonella spp. and Shigella spp. in milk.</a> Int J Food Microbiol. 148 (2), 75-79 (2011).</li><li>Fang, X., Chen, H., Yu, S., Jiang, X., Kong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+viruses+accurately+by+a+multiplex+microfluidic+loop-mediated+isothermal+amplification+chip.">Predicting viruses accurately by a multiplex microfluidic loop-mediated isothermal amplification chip.</a> Anal Chem. 83 (3), 690-695 (2011).</li><li>Stedtfeld, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene-Z:+a+device+for+point+of+care+genetic+testing+using+a+smartphone.">Gene-Z: a device for point of care genetic testing using a smartphone.</a> Lab Chip. 12 (8), 1454-1462 (2012).</li><li>Liu, D., Liang, G., Zhang, Q., Chen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Mycobacterium+tuberculosis+using+a+capillary-array+microsystem+with+integrated+DNA+extraction,+loop-mediated+isothermal+amplification,+and+fluorescence+detection.">Detection of Mycobacterium tuberculosis using a capillary-array microsystem with integrated DNA extraction, loop-mediated isothermal amplification, and fluorescence detection.</a> Anal Chem. 85 (9), 4698-4704 (2013).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point-of-Care+Multiplexed+Assays+of+Nucleic+Acids+Using+Microcapillary-based+Loop-Mediated+Isothermal+Amplification.">Point-of-Care Multiplexed Assays of Nucleic Acids Using Microcapillary-based Loop-Mediated Isothermal Amplification.</a> Anal Chem. 86 (14), 7057-7062 (2014).</li><li>Shao, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+detection+of+multiple+genetically+modified+organisms+in+a+capillary+array.">Visual detection of multiple genetically modified organisms in a capillary array.</a> Lab Chip. 17 (3), 521-529 (2017).</li><li>Lizardi, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+detection+and+single-moledule+counting+using+isothermal+rolling-circle+amplification.">Mutation detection and single-moledule counting using isothermal rolling-circle amplification.</a> Nat Genet. , (1998).</li><li>Piepenburg, O., Williams, C. H., Stemple, D. L., Armes, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+detection+using+recombination+proteins.">DNA detection using recombination proteins.</a> Plos Biol. 4 (7), 1115-1121 (2006).</li><li>Opota, O., Jaton, K., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+diagnosis+of+bloodstream+infection:+towards+molecular+diagnosis+directly+from+blood.">Microbial diagnosis of bloodstream infection: towards molecular diagnosis directly from blood.</a> Clin Microbiol Infect. 21 (4), 323-331 (2015).</li></ol>

作者没有什么可透露的。

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>MnCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>MKBP0495V</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</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 ...

visual-detection-of-multiple-nucleic-acids-in-a-capillary-array

Research

JoVE Journal

Bioengineering

7.2K 次观看.  Shanghai Jiao Tong University. 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。

视频: Visual Detection of Multiple Nucleic Acids in a Capillary Array

The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. This approach to colorimetric DNA detection was based on the hybridization-mediated growth of gold nanoparticle probes (AuNP probes). The growth size and configuration of the AuNP are dominated by the number of DNA samples hybridized with the probes. Based on the specific size- and shape-dependent optical properties of the nanoparticles, the number of mismatches in a sample DNA fragment to the probes is able to be discriminated. The tests were conducted via droplets containing reagents and DNA samples respectively, and were transported and mixed on the pneumatic platform with the controlled pneumatic suction of the flexible PDMS-based superhydrophobic membrane. Droplets can be delivered simultaneously and precisely on an open-surface on the proposed pneumatic platform that is highly biocompatible with no side effect of DNA samples inside the droplets. Combining the two proposed methods, the multi-nucleotide polymorphism can be detected at sight on the pneumatic droplet manipulation platform; no additional instrument is required. The procedure from installing the droplets on the platform to the final result takes less than 5 min, much less than with existing methods. Moreover, this combined MNP detection approach requires a sample volume of only 10 &#181;l in each operation, which is remarkably less than that of a macro system.

This work presents a simple and visual method to detect multi-nucleotide polymorphisms on a pneumatic droplet manipulation platform. With the proposed method, the entire experiment, including droplet manipulation and detection of multi-nucleotide polymorphisms, can be performed near 23 &#176;C without the aid of advanced instruments.

在气动操纵液滴平台多核苷酸多态性的视觉比色法检测

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. ...

科研

遗传学

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

ampliPHOX比色法检测为流感的DNA芯片

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

免疫与感染

Real-time Tracking of DNA Fragment Separation by Smartphone

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

通过智能手机实时跟踪DNA片段分离

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

生物化学

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications.

This protocol outlines the fabrication of a large-scale, multiplexed two-dimensional DNA or antibody array, with potential applications in cell signaling studies and biomarker detection.

高密度条码抗体芯片的水流模式的指导下制作

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this ...

化学

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

生物工程

<meta charset="utf8"></head><body>用于鉴别性 RNA 和 DNA 分析的精密 DNAzyme 纳米机器

DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their functions include tight analyte binding, highly selective analyte recognition, fluorescent signal amplification by multiple catalytic cleavages of a fluorogenic reporter substrate, and fluorogenic substrate attraction for an increase in sensor response. Functional units are attached to a common DNA scaffold for their cooperative action. The RNA-cleaving 10-23 DNMs feature&#160;improved sensitivity in comparison with non-catalytic hybridization probes. The stability of the DNM and the increased chances of substrate recognition are provided&#160;by a double-stranded DNA fragment,&#160;a tile. DNM can differentiate two analytes with a single nucleotide difference in a folded RNA and a double-stranded DNA and detect analytes at concentrations ~1000 times lower than other protein-free hybridization probes. This article presents the concept behind the diagnostic potential of DNA-nanomachine activity and overviews DNM design, assembly, and application&#160;in nucleic acid detection assays.

<meta charset="utf8"></head><body>作者聚焦:DNA 纳米传感器的进步 – 解决分子检测中的灵敏度和选择性挑战

The DNAzyme-based nanomachines can be used for highly selective and sensitive detection of nucleic acids. This article describes a detailed protocol for the design of DNAzyme-based nanomachines with a 10-23 core using free software and their application in the detection of an Epstein-Barr virus fragment as an example.

DNAzyme 10-23 - 用于核酸识别的基于纳米机器

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their ...

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

单细胞多重荧光成像，可可视化病毒核酸和蛋白质，并监测 HIV、HTLV、HBV、HCV、寨卡病毒和流感感染

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

生物学

Capillary Electrophoresis: A Technique to Analyze Fluorescently Labeled Polymerase Chain Reaction Amplicons

Source: Gulla, S. et al., <a href="https://www.jove.com/v/59455">Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis</a>. J. Vis. Exp. (2019)
This video describes the technique of capillary electrophoresis to analyze fluorescently labeled PCR amplicons obtained from various samples based on their size. It generates a migratory pattern that determines the fragments&#8217; base-pair lengths with the help of the labeled fluorescent dye in each fragment.

毛细管电泳:一种分析荧光标记的聚合酶链反应扩增子的技术

Source: Gulla, S. et al., Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and ...

毛细管阵列中多核酸的视觉检测

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微流体为基础的电化学生物芯片的无标记的DNA杂交分析

DNAzyme 10-23 - 用于核酸识别的基于纳米机器

DNAzyme 10-23 - 用于核酸识别的基于纳米机器

DNAzyme 10-23 - 用于核酸识别的基于纳米机器

单细胞多重荧光成像，可可视化病毒核酸和蛋白质，并监测 HIV、HTLV、HBV、HCV、寨卡病毒和流感感染

毛细管电泳:一种分析荧光标记的聚合酶链反应扩增子的技术