This protocol development was supported by the Bill and Melinda Gates Foundation Grand Challenges in Global Health grant 37774. Real-time PCR reagents and advice were provided by Abbott Molecular Inc. Des Plaines, IL. 8E5-LAV cells were provided by the Virology Quality Assurance Laboratory, Rush Presbyterian; St. Luke's Medical Center. HIV-1 negative blood was provided by Core Lab, NorthShore University HealthSystems, Evanston, IL. Thanks to Mark Fisher for photography help.

伦理声明：在本研究中使用的全血标本不被认为是涉及人类受试者的研究。用于临床诊断目的，这是满足了获得的样本，并为FINA研究测定提供这些试样的剩余部分。试样进行编码，使得研究者不能够容易地确定个人的身份。 1.国际泳联样品制备模块的研制<ol><li>准备用切割35×35毫米2.6 平方毫米厚的100％化妆棉吸水垫。切标本每一个焊盘。 </li><li>制备塑料石蜡膜与纸背衬带通过切?...

<ol>
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EID联系到快速治疗的访问已被证明是降低婴儿死亡率由于HIV感染16。由于婴儿的血液母源抗体的持久性，快速的艾滋病病毒抗体测试已确定HIV暴露婴儿的状态用处有限。世界卫生组织建议，出生HIV-1阳性母亲的婴儿都应该在4-6周龄用病毒学测试17进行测试。我们已经报道HIV-1的原病毒DNA的全血标本中检测和定量，能够单拷贝检测与证实100％的敏感性和特异性的61的中国婴儿11...

若干报告已经与使精美灵敏度和分子诊断的特异性可用于所有的目的讨论了点- -关心（POC）装置1-5的造纸或膜-基于提取方法中使用的开发。世界卫生组织（WHO）性传播疾病诊断倡议创造ASSURED（价格实惠，敏感，特异，用户界面​​友好，快速和强大，装备自由并交付给那些谁需要它）来描述理想的POC的特征词测试6。这些准则的免费装备，特点是特别具有挑战性，以实现分子诊断。然而，在该领域的每一次创新将推动实现这些最需要的目标，并有通过调整现有的技术7是希望短期内改善测试性能。 这里介绍一个简单的协议，用于从全血中提取DNA不需要复杂的化学或实验室仪器。国际泳联（核酸过滤隔离）的样品制备方法最初是提取全血白细胞DNA，以检测HIV-1前病毒定量PCR样品到答案点保健（POC）的一部分（定量PCR）婴儿早期诊断（EID）平台，在资源有限的设置8-11使用。 FINA提取不同于其中使用二氧化硅膜或氧化硅-包被的顺颗粒在离液剂12的存在下可逆地结合DNA常规纯化方法。相反，国际泳联采用立式过滤通过分离膜直接提取全血细胞DNA。含有包埋的DNA的膜可以直接在PCR管放置并且立即在购买扩增9干燥的PCR反应或空气中。没有离液剂，苯酚或醇是在样品提取使用，elimina婷去除提取过程13,14衍生有力的qPCR抑制剂需要大量洗涤步骤。 国际泳联膜可以捕获样品被添加到膜之前通过细胞裂解11解放或细胞9或基因组DNA。用于小区捕获，全血直接加入到该膜。细胞随后在膜中加入10毫摩尔的NaOH裂解。这种方法的优点是，它仅涉及3个步骤：1）加入样品; 2）细胞裂解/洗3）过滤盘放置在定量PCR管。这种方法的缺点是，该膜只能容纳一个定义单元成正比的膜盘的直径的数量。为了达到为EID所需的检测的限制，将100μl的全血需要这需要一个过滤器，过大，被放置在qPCR的管样品输入。裂解血细胞用洗涤剂添加样品到集合前膜增加了一个处理步骤，但允许对同一样本大小使用较小的滤波器。我们能够证明高再现性，单拷贝检测，并从100微升的血液使用这种测试配置11的HIV-1原病毒DNA的少至10个拷贝的定量。 在这份报告中，我们描述最初实验室使用开发的国际泳联协议。称为FINA样品制备模块的膜/滤膜夹层可以大批量制备，并储存起来以备后用。当试样被提取这个过程需要2分钟，并可以在不同大小的批次进行。在定量PCR可立即运行或包含嵌入DNA中的过滤器可以被存储直到它方便执行定量PCR。这种方法是用于在低和高资源设置标本常规分析非常方便。

<table border="0" cellpadding="0" cellspacing="0" width="860">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Fusion 5</td>
			<td style="width:179px;">GE Healthcare Life Sciences</td>
			<td style="width:104px;">8151-9915</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">707 blotting pad</td>
			<td>VWR International</td>
			<td>28298-014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PARAFILM M</td>
			<td>VWR International</td>
			<td>52858-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3M Double-Coated Polyester Diagnostic Tape&#160;</td>
			<td>3M Medical Specialties</td>
			<td>9965</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 &#181;l qPCR strip tubes</td>
			<td>Agilent</td>
			<td>401428</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">optical strip caps</td>
			<td>Agilent</td>
			<td>401425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mx3005p qPCR System</td>
			<td>Agilent</td>
			<td>401456</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td>A.C.S. Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td>BioXtra</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td>for molecular biology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fetal bovine serum, certified, U.S. origin</td>
			<td>Thermo Fisher Scientific</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hammer driven small hole punch 3/16&#34; hole diameter (5.1 mm)</td>
			<td>McMaster Carr</td>
			<td>3424A16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hammer driven small hole punch 1/4&#34; hole diameter (7.14 mm)</td>
			<td>McMaster Carr</td>
			<td>3424A19</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hammer driven small hole punch 5/16&#34; hole diameter (8.35 mm)</td>
			<td>McMaster Carr</td>
			<td>3424A23</td>
			<td></td>
		</tr>
	</tbody>
</table>

filtration isolation of nucleic acids:  a simple and rapid dna extraction method

FINA，核酸过滤分离，是通过分离膜和吸收垫利用垂直过滤提取从全血细胞的DNA，在不到2分钟的新颖的提取方法。血液检体与洗涤剂，短暂混合处理，并用移液管施加到分离膜。溶解物芯吸入印迹垫由于毛细管作用，捕捉分离膜的表面上的基因组DNA。期间的简单的洗涤步骤，其中PCR抑制剂是恶进入吸湿印迹垫提取的DNA被保留在膜上。然后将含有包埋的DNA的膜被加入到没有进一步纯化的PCR反应。这种简单的方法不需要实验室设备，并可以用廉价实验室用品很容易地实现。在这里，我们描述了从100微升全血中HIV-1前病毒DNA的高度敏感的检测和定量的协议作为一种模式在早期艾滋病毒的FANT诊断，可以容易地适用于其他遗传靶。

用于提取从8E5-LAV细胞掺入的全血前病毒DNA的工作流程示于图1， 图2示出了FINA示例模块和制备的qPCR管。这种方法允许从8E5-LAV细胞以不同拷贝数的HIV-1原病毒的有效扩增，如图做作标本（ 图3）的标准曲线。 PCR具有103％的效率，如从效率= 1 + 10（-1 /斜率）计算。线的方程为y = -3.25x + 27.95，与R 2 = 0.996的相关性。艾滋病毒原病毒DNA...

Watch this Scientific Journal Video about Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method at JoVE.com

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

在这里，我们描述了一个简单，快速的基于纸张的DNA提取的HIV前病毒DNA的全血通过定量PCR检测方法。这种协议可以在检测其他遗传标记或使用替代的扩增方法被扩展为使用。

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