Name: rapid, safe, and simple manual bedside nucleic acid extraction for the detection of virus in whole blood samples
Uploaded: 2018-06-30T15:00:00
Description: Watch this Scientific Journal Video about Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples at JoVE.com

我们感谢 Susanne. 拉斯穆森和 Solvej Kolbjørn 詹森为他们的技术援助和处理临床样品。该项目是 EbolaMoDRAD 财团的一部分, 该集团已从创新医学倡议2根据赠款协议 N°115843的联合承诺获得资金。这项联合承诺得到欧洲联盟2020地平线研究和创新计划和 EFPIA 的支持。

资本区域生物医学研究伦理学委员会已给予知情同意, 此处描述的所有方法均获豁免, 不受道德委员会制度的审查, 这是根据丹麦关于化验发展项目的法律。
1. 用于病毒灭活和快速 NA 提取的采血真空管的研制
注意: 此协议使用的缓冲区包含胍硫氰酸盐 (GITC) 和非离子表面活性剂, 这是刺激物。采取适当的实验室安全措施, 使用流动罩, 并在处理时戴上手套。避...

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在本报告中, 我们描述了一种安全、快速、简单的人工床边 NA 提取方法, 用于在裂解/捆绑缓冲-灭活全血中对病毒进行下游分子检测。所述的 NA 提取方法是直接对含有裂解/捆绑缓冲 (材料表) 的真空采血管收集的全血标本进行的.7。此特定缓冲区钝化 EBOV7 , 是该方法中的关键组件。血液收集后, 我们建议孵化管至少20分钟7 , 以确保完...

在病毒暴发的情况下, 当病人被限制在医院隔离病房或需要快速诊断时, 安全、简单、准确的点对诊的分子诊断是必要的病人护理和风险遏制。最近两次病毒暴发, 在西非的埃博拉病毒 (EBOV) 和南美的 Zika 病毒 (2015), 增加了对改进的护理点分子诊断测试的兴趣, 如反向转录回路介导的等温放大 (RT 灯)1,2和 recombinase 聚合酶放大 (RPA)3,4。RT 灯和 RPA 都是快速、敏感和特定的分子测试, 可以在简化的样品制备中进行。对 Zika 病毒, 将 RT 灯与侧流法 (LFA) 相结合, 在30分钟内检测非纯化全血标本中的 Zika 病毒;然而, 对于被归类为风险组4病原体并具有高度传染性的 EBOV, 需要在生物安全等级 4 (BSL-4) 条件下处理和灭活, 然后才能进行任何安全诊断程序。
EBOV 的简化失活方法, 例如在2、3、4、5的样品中加入裂解缓冲剂, 在暴发期间使用;然而, 这些方法需要在 BSL-4 条件下处理与实验室设备, 如 BSL-3 生物安全柜, 离心机, 暖气块, 吸管, 至少。这种设备通常不存在于隔离病房或外地医院。为了克服这一挑战, 已尝试在手提箱3中进行诊断, 并开发了一些便携设备和机器 (例如, 一种用于核酸 (NA) 提取的便携设备)6。然而, 在使用这些设备之前, EBOV 阳性样品仍需进行灭活。
我们以前报告了一个快速的床边病毒灭活方法为 EBOV7, 痘苗病毒和牛痘病毒8通过增加一个商业裂解/捆绑缓冲到普通的真空采血管, 允许直接血液从患者转移到灭活缓冲器7。这种直接和直接的钝化在一个封闭的系统, 消除了处理样品使用任何严格的遏制, 如 BSL-4 条件7, 并在正常 BSL-2 条件下处理样品的需要。这种灭活方法与几种 NA 萃取系统兼容, 如机器人和手净化套件7;然而, 这些方法需要实验室设备, 如机器人, 离心机, 和电力, 这并不总是存在于现场设置或在医院隔离病房。
在本报告中, 我们描述了一种安全、快速和简化的人工 NA 提取方法, 用于随后在裂解/捆绑缓冲-灭活全血中的病毒分子检测。NA 萃取方法不需要任何设备, 除了磁铁/磁性持有人。NA 萃取不需要离心机、加热块或电。因此, 这种方法不依赖于实验室设施, 而且可以很容易地用于任何地方 (如在野战医院、医院隔离病房或低资源环境下)。na 萃取方法快速简便, 可直接用于任何下游 NA 试验, 如 qPCR、rt qPCR、灯或 rt 灯。当该 NA 提取方法与灯和便携式电池驱动的等温仪相结合时, 可以在40分钟内采集到血液中的床边诊断。

<table border="0" cellpadding="0" cellspacing="0" style="width:1080px;" width="1081">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:544px;">Vacutainer K2 EDTA tubes (4 mL)</td>
			<td style="width:251px;">Becton Dickinson</td>
			<td style="width:119px;">368861</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plus Blood Collection</td>
			<td>Becton Dickinson</td>
			<td style="width:119px;">362725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">23G x 1 needle&#160;</td>
			<td>Becton Dickinson</td>
			<td style="width:119px;">300800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LUER LOK syringe (3 mL)</td>
			<td>Becton Dickinson</td>
			<td style="width:119px;">309658</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Butterfly needle with small-bore extension tubing</td>
			<td style="width:251px;">J&#248;rgen Kruuse A/S</td>
			<td style="width:119px;">121714</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1% Virkon&#160;</td>
			<td>Nomeco A/S</td>
			<td style="width:119px;">849265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MagNA Pure LC DNA Isolation Kit I - Lysis/Binding Buffer - Refill</td>
			<td>Roche Diagnostics A/S</td>
			<td style="width:119px;">03246752001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MagNA Pure LC Total Nucleic Acid Isolation Kit</td>
			<td>Roche Diagnostics A/S</td>
			<td style="width:119px;">3038505001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc&#8482; Biobanking and Cell Culture Cryogenic Tubes (1,8 mL)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">375418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc&#8482; Biobanking and Cell Culture Cryogenic Tubes (3,6 mL)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">379189</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc&#8482; Biobanking and Cell Culture Cryogenic Tubes (4,5 mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">379146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DynaMag&#8482;-5 Magnet</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">12303D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer pipette (3.5 mL)</td>
			<td>Sarstedt</td>
			<td style="width:119px;">86.1171.010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Scientific&#8482; Samco&#8482; Fine Tip Transfer Pipettes (1.5 mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">231</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Thermo Scientific&#8482; Nunc&#8482; 50 mL Conical Sterile Polypropylene Centrifuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">339652</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid, safe, and simple manual bedside nucleic acid extraction for the detection of virus in whole blood samples

对感染的快速诊断对于暴发管理、风险控制和病人护理至关重要。我们以前已经显示了一种方法, 快速床边的埃博拉病毒在血液取样的安全核酸 (NA) 测试通过添加一个商业裂解/捆绑缓冲直接进入真空采血管。利用这种床边灭活方法, 我们开发了一种安全、快速、简便的床边 NA 提取方法, 用于随后在裂解/捆绑缓冲-灭活全血中检测病毒。钠萃取直接在采血管中进行, 无需设备或电。
将血液收集到溶解/结合缓冲液中后, 通过手工翻转管将其内容混合, 并在室温下孵化出20分钟的混合物。将磁性玻璃微粒 (MGPs) 添加到管中, 通过手工翻转收集管将其内容混合。MGPs, 然后收集在血液收集管的一侧使用磁性持有人或磁铁和橡皮筋。MGPs 清洗三次, 在将洗脱缓冲器直接添加到采集管后, NAs 就可以进行 NA 测试, 如 qPCR 或等温环放大 (灯管), 而不需要从反应中去除 MGPs。NA 提取方法不依赖任何实验室设施, 可以很容易地在任何地方使用 (例如, 在外地医院和医院隔离病房)。当该 NA 提取方法与灯和便携式仪器相结合时, 可在采血40分钟内获得诊断。

本文提出的协议简单有效, 可广泛应用于用裂解/结合缓冲剂灭活的传染性全血标本进行的任何分子检测。血液失活和 NA 提取工作流程如图 1所示, 包括采血真空管的制备7 (图 1A), 采血7 (图 1B), NA 提取 (图 1C - 1H) 和下游 NA 分析 (图 ...

Watch this Scientific Journal Video about Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples at JoVE.com

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

在这里, 我们提出了一个快速病毒核酸提取从病毒灭活全血的协议。萃取是直接在采血管, 不需要设备或电力。该方法不依赖于实验室设施, 可以在任何地方使用 (例如在野战医院)。

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 ...

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