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.

Ethics statement: The whole blood specimens used in this study are not considered to be research involving human subjects. The specimens were obtained for clinical diagnostic purposes, which were satisfied, and the remaining portion of these specimens was provided for the FINA research assay. The specimens were coded such that the investigators were not able to readily ascertain the identity of individuals.
1. Preparation of FINA Sample Preparation Module
<ol>
	<li>Prepare blotting pad by cutting a 35 x 35 mm<su...

<ol>
	<li>Byrnes, S. A., 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=One-step+purification+and+concentration+of+DNA+in+porous+membranes+for+point-of-care+applications.">One-step purification and concentration of DNA in porous membranes for point-of-care applications.</a> Lab Chip. 15 (12), 2647-2659 (2015).</li><li>Connelly, J. T., Rolland, J. P., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Paper+Machine"+for+Molecular+Diagnostics.">"Paper Machine" for Molecular Diagnostics.</a> Anal Chem. 87 (15), 7595-7601 (2015).</li><li>Govindarajan, A. V., Ramachandran, S., Vigil, G. D., Yager, P., Bohringer, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low+cost+point-of-care+viscous+sample+preparation+device+for+molecular+diagnosis+in+the+developing+world;+an+example+of+microfluidic+origami.">A low cost point-of-care viscous sample preparation device for molecular diagnosis in the developing world; an example of microfluidic origami.</a> Lab Chip. 12 (1), 174-181 (2012).</li><li>Linnes, J. 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=Paper-based+molecular+diagnostic+for.">Paper-based molecular diagnostic for.</a> RSC Adv. 4 (80), 42245-42251 (2014).</li><li>Rodriguez, N. 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=Paper-Based+RNA+Extraction,+in+Situ+Isothermal+Amplification,+and+Lateral+Flow+Detection+for+Low-Cost,+Rapid+Diagnosis+of+Influenza+A+(H1N1)+from+Clinical+Specimens.">Paper-Based RNA Extraction, in Situ Isothermal Amplification, and Lateral Flow Detection for Low-Cost, Rapid Diagnosis of Influenza A (H1N1) from Clinical Specimens.</a> Anal Chem. 87 (15), 7872-7879 (2015).</li><li>Peeling, R. W., Holmes, K. K., Mabey, D., Ronald, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+tests+for+sexually+transmitted+infections+(STIs):+the+way+forward.">Rapid tests for sexually transmitted infections (STIs): the way forward.</a> Sex Transm Infect. 82, 1-6 (2006).</li><li>Mabey, D., Peeling, R. W., Ustianowski, A., Perkins, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostics+for+the+developing+world.">Diagnostics for the developing world.</a> Nat Rev Microbiol. 2 (3), 231-240 (2004).</li><li>Jangam, S. R., Agarwal, A. K., Sur, K., Kelso, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+point-of-care+PCR+test+for+HIV-1+detection+in+resource-limited+settings.">A point-of-care PCR test for HIV-1 detection in resource-limited settings.</a> Biosens Bioelectron. 42, 69-75 (2013).</li><li>Jangam, S. R., Yamada, D. H., McFall, S. M., Kelso, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+point-of-care+extraction+of+human+immunodeficiency+virus+type+1+proviral+DNA+from+whole+blood+for+detection+by+real-time+PCR.">Rapid point-of-care extraction of human immunodeficiency virus type 1 proviral DNA from whole blood for detection by real-time PCR.</a> J Clin Microbiol. 47 (8), 2363-2368 (2009).</li><li>Kelso, D. M., Jangam, S., Yamada, D., McFall, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Google Patents. , (2012).</li><li>McFall, S. 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=A+Simple+and+Rapid+DNA+Extraction+Method+from+Whole+Blood+for+Highly+Sensitive+Detection+and+Quantitation+of+HIV-1+Proviral+DNA+by+Real-Time+PCR.">A Simple and Rapid DNA Extraction Method from Whole Blood for Highly Sensitive Detection and Quantitation of HIV-1 Proviral DNA by Real-Time PCR.</a> Journal of Virological Methods. 214, 37-42 (2015).</li><li>Boom, R., 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=Rapid+and+simple+method+for+purification+of+nucleic+acids.">Rapid and simple method for purification of nucleic acids.</a> J Clin Microbiol. 28 (3), 495-503 (1990).</li><li>Radstrom, P., Knutsson, R., Wolffs, P., Lovenklev, M., Lofstrom, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-PCR+processing:+strategies+to+generate+PCR-compatible+samples.">Pre-PCR processing: strategies to generate PCR-compatible samples.</a> Mol Biotechnol. 26 (2), 133-146 (2004).</li><li>Schrader, C., Schielke, A., Ellerbroek, L., Johne, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR+inhibitors+-+occurrence,+properties+and+removal.">PCR inhibitors - occurrence, properties and removal.</a> J Appl Microbiol. 113 (5), 1014-1026 (2012).</li><li>Folks, T. M., Powell, D. M., Martin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Google Patents. , (1991).</li><li>Violari, A., 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=Early+Antiretroviral+Therapy+and+Mortality+among+HIV-Infected+Infants.">Early Antiretroviral Therapy and Mortality among HIV-Infected Infants.</a> N Engl J Med. 359 (21), 2233-2244 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> World Health Organization. , (2010).</li><li>Gruner, N., Stambouli, O., Ross, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dried+blood+spots--preparing+and+processing+for+use+in+immunoassays+and+in+molecular+techniques.">Dried blood spots--preparing and processing for use in immunoassays and in molecular techniques.</a> J Vis Exp. (97), (2015).</li><li>Maiers, T. J., 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=An+investigation+of+fingerstick+blood+collection+for+point-of-care+HIV-1+viral+load+monitoring+in+South+Africa.">An investigation of fingerstick blood collection for point-of-care HIV-1 viral load monitoring in South Africa.</a> S Afr Med J. 105 (3), 228-231 (2015).</li></ol>

The authors have nothing to disclose.

EID linked to rapid treatment access has been demonstrated to reduce infant mortality due to HIV infection 16. Because of the persistence of maternal antibodies in an infant&#39;s blood, rapid HIV antibody tests have limited utility in determining the status of HIV-exposed infants. The WHO recommends that all infants born to HIV-1 positive mothers should be tested at 4-6 weeks of age, using a virological test 17. We have reported the development of an assay for the detection and quantitation of HIV-...

Several reports have discussed the development of paper- or membrane-based extraction methods for use in point-of-care (POC) devices 1-5 with the aim of making the exquisite sensitivity and specificity of molecular diagnostics available to all. The World Health Organization (WHO) Sexually Transmitted Diseases Diagnostics Initiative coined the term ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free and Delivered to those who need it) to describe the ideal characteristics of a POC test 6. Of these guidelines, the equipment-free characteristic is particularly challenging to achieve for molecular diagnostics. However, every innovation in the field will advance the goal of reaching those in most need, and there is hope for near-term improvements in test performance by adapting existing technology 7.
Here we describe a simple protocol for extracting DNA from whole blood that does not require complex chemistry or laboratory instrumentation. The FINA (filtration isolation of nucleic acids) sample preparation method was originally developed to extract leukocyte DNA from whole blood in order to detect the HIV-1 provirus as part of a sample-to-answer point-of-care (POC) quantitative PCR (qPCR) early infant diagnostic (EID) platform for use in limited resource settings 8-11. FINA extraction differs from conventional purification methods which use silica membranes or silica-coated paramagnetic particles to reversibly bind DNA in the presence of chaotropic agents 12. Instead, FINA uses vertical filtration via a separation membrane to extract cellular DNA from whole blood directly. The membrane containing the entrapped DNA can be placed directly in a PCR tube and either immediately used in a PCR reaction or air dried for later amplification 9. No chaotropic agents, phenol, or alcohols are used in the sample extraction, eliminating the extensive washing steps needed to remove potent qPCR inhibitors derived from the extraction process 13,14.
The FINA membrane can capture either cells 9 or genomic DNA liberated by cell lysis 11 before the specimen is added to the membrane. For the cell capture, whole blood is added directly to the membrane. The cells are subsequently lysed in the membrane by adding 10 mM NaOH. The advantage to this method is that it involves only 3 steps: 1) sample addition; 2) cell lysis/wash and 3) filter disk placement in qPCR tube. The drawback to this method is that the membrane can only hold a defined number of cells directly proportional to the diameter of the membrane disk. To reach the limit of detection required for EID, 100 &#181;l of whole blood is required for sample input which entails a filter that is too large to be placed in a qPCR tube. Lysing the blood cells with detergent before adding the sample to the collection membrane adds a processing step, but allows the use of a smaller filter for the same sample size. We were able to demonstrate high reproducibility, single copy detection, and quantification of as little as 10 copies of HIV-1 proviral DNA from 100 &#181;l of blood using this test configuration 11.
In this report, we describe the FINA protocol as originally developed for laboratory use. The membrane/filter sandwich known as the FINA sample preparation module can be prepared in large batches and stockpiled for later use. When specimens are to be extracted this process takes 2 min and can be performed in varying size batches. The qPCR can be run immediately or the filters containing the embedded DNA can be stored until it is convenient to perform qPCR. This method is very convenient for routine analysis of specimens in both low and high resource settings.

<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, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

The workflow for extracting proviral DNA from whole blood spiked with 8e5-LAV cells is shown in Figure 1. Figure 2 shows the FINA sample module and prepared qPCR tube. This method allows for efficient amplification of HIV-1 provirus from 8e5-LAV cells at different copy numbers, as shown in the standard curve of contrived specimens (Figure 3). PCR had an efficiency of 103%, as calculated from Efficiency = -1+10(-1/slope). Equati...

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

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

The overall goal of this FINA method which stands for filtration isolation of nucleic acids is to isolate genomic DNA for PCR amplification from complex specimens quickly and simply without the use of laboratory instrumentation such as a centrifuge. This rapid paper based DNA extraction method for HIV pro viral DNA was developed for incorporation into a quantitative PCR diagnostic device. The main advantage of this technique is that the sample prep modules and PCR tubes can be prepared in advance so the DNA extraction takes less than two minutes.We first developed this method to be incorporated into a sample to answer early infant diagnostic test for HIV-1 from heelstick blood. Generally individuals new to this method may struggle because removing the backing of the double sided tape that holds the cell capture membrane to the side of the PCR tube requires practice. Demonstrating the procedure will be Jen Reed, a senior technician from my lab.Begin this procedure by preparing the materials needed for assembling the FINA sample preparation module. Prepare the blotting pad by cutting a 35 by 35 millimeter square of a 2.6 millimeter thick 100%cotton pad. Prepare plastic paraffin film with a paper backing tape.Cut a 25 by 50 mm piece of paraffin film and then use a hammer to punch a 7.14 mm diameter hole in the center of the tape. Prepare a bound glass blood separator membrane or capture disk by using a hammer to punch an 8.35 mm diameter circle. To assemble the FINA sample preparation module use forceps to place the capture disc in the center of the blotting pad.Remove the backing of the paraffin film tape and place it over the capture disc so that the whole of the paraffin tape leaves the center of the capture disc exposed. Ensure maximum contact between the disc and the blotting pad by stretching the paraffin tape slightly to cover the blotting pad and pressing the paraffin tape firmly to stick it to the blotting pad around all edges of the disc. A 5.1 mm tape disc is required to anchor the capture membrane to the side of qPCR tube to prevent the membrane from blocking the fluorescence detection of the real time PCR instrument.Prepare this tape disc by using a hammer to punch a circle of double coated polyester diagnostic tape from a sheet of the tape. Using forceps remove one side of the tape sticker liner. Place the tape into a 200 microliter PCR tube, sticking it onto one side and toward the bottom of the tube.Remove the second sticker liner. The tube is now ready to receive the prepared disc. The detection of proviral DNA from whole blood spiked with 8e5 LAV cells will be demonstrated in this video.Thaw out the previously frozen 8e5 LAV cell pellets on ice. Prepare a serial dilution of the cells in freezing medium with concentrations ranging from 1 to 400 cells per microliter. Pipette 10 microliters of cells from each of the dilutions into a micro centrifuge tube.Add 100 microliters of fresh HIV-1 negative eDTA treated whole blood sample to each tube to create a standard curve. Mix by gently flicking the bottom of the tube five times. To begin the DNA extraction procedure, lyse the blood cells by adding triton X-100 to a final concentration of one percent.Mix by gently flicking the bottom of the tube five times. The blood should turn a translucent red. Pipette the entire blood specimen onto the capture disc.A water tight seal between the paraffin tape and the capture membrane ensures that the blood flows through the membrane. And good contact between the contact membrane and blotting pad assembly ensures rapid sample wicking. Allow the sample to soak through the filter.The blood lysate has soaked into the capture disc and the disc appears mat. Add 600 to 1, 000 microliters of 10 mmol sodium hydroxide dropwise onto the capture disc. The filter will change from red to white, indicating clearance of hemoglobin.Next separate the disc from the blotting pad and apply the disc to the tape in the prepared PCR tube. After the filter is placed in the PCR tube analyze the sample right away. Prepare the qPCR master mix as described in the protocol text and be sure that the filter is completely covered with the master mix and stays fixed to the side of the tube throughout the reaction.Perform amplification using a real time PCR device as described in the protocol text. Alternatively if qPCR cannot be performed immediately dry the filter overnight in a box containing calcium sulfate dessicant. On the following day, cap and place the PCR tube in a foiled pouch with silica dessicant and store at room temperature until ready for analysis.This method allows for efficient amplification of HIV-1 provirus from 8e5 LAV cells at different copy numbers. The solid lines are amplification plots obtained from four replicates of HIV-1 negative whole blood spiked with 4, 000, 400, 40 and 10 8e5 LAV cells with 500 copies per reaction of the internal control. The dotted line indicates the threshold.This next graph shows standard curves of quantitation cycle values calculated from the amplification plot vs. log copy number of 8e5 LAV cells per 100 microliters of whole blood. The calculated PCR efficiency was 103%The HIV proviral DNA standard curve replicates also have highly reproducible amplification.Additionally the presence of an internal control of 500 copies of hydroxy pyruvate reductase shows that there is no PCR inhibition resulting from extracting blood with this method independent of the number of copies of HIV-1 provirus present. The solid lines represent the amplification plots and the dotted line represents the threshold. Following this procedure PCR targets can be amplified from various specimen types to detect your gene of interest.For example cheek swabs for pharmacogenetic screening or blood for rapid genotyping of animal models. Don't forget that working with blood borne pathogens can be extremely hazardous and universal precautions should always be taken while performing this procedure.

filtration-isolation-nucleic-acids-simple-rapid-dna-extraction

Research

JoVE Journal

Biology

17.9K Görüntüleme.  Northwestern University. The overall goal of this FINA method which stands for filtration isolation of nucleic acids is to isolate genomic DNA for PCR amplification from complex specimens quickly and simply without the use of laboratory instrumentation such as a centrifuge. This rapid paper based DNA extraction method for HIV pro viral DNA was developed for incorporation into a quantitative PCR diagnostic device. The main advantage of this technique is that the sample prep modules and PCR tubes can be prepared in adv...