a technique for magnetic glass particle-mediated nucleic acid extraction from blood

A Technique for Magnetic Glass Particle-Mediated Nucleic Acid Extraction from Blood

To purify nucleic acids from the collected blood, again mix the contents of the tube by flipping the vacuum tube by hand 5 to 10 times. Pour the prepared aliquot of MGPs directly into the blood collection tube, then, place a new lid from an unused blood collection tube on the tube containing the sample. Place a finger on the lid to ensure that the tube is tightly closed, and mix the contents of the tube by flipping 5 to 10 times.
Place the tube in the magnetic holder and keep a finger on the lid to ensure the tube is tightly closed. Then, flip the magnetic holder with the tube a few times to disperse the MGPs before they collect on the side of the tube with the magnet. Remove the lid of the tube and discard the contents of the tube into a 50-milliliter collection tube using a disposable pipette.
Pour the prepared aliquot of WB1 directly into the blood collection tube, and close the lid on the tube. Mix the content by hand 5 to 10 times. Then, collect the beads at the side of the tube using the magnet, and remove the liquid as before. Resuspend the beads in the prepared aliquot of WB2. Then, discard the solution, and repeat the procedure with the prepared aliquot of WB3.
Finally, remove the tube from the magnetic holder and pour the prepared aliquot of EB directly into the blood collection tube. Place the lid on the tube, and resuspend the MGPs by tapping 5 to 10 times with a finger. Finally, use a 1.5-milliliter disposable pipette to transfer one droplet of the resuspended MGPs to the downstream nucleic acid amplification reaction mix.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines
1. Blood Collection from Patients
CAUTION: Take appropriate laboratory safety measures when collecting whole blood from the patient. Wear gloves and glasses. If the patient is in isolation, please follow biosafety level 4 procedures.
<ol>
	<li>To collect intravenous whole blood from the patient, use a butterfly needle with small-bore extension tubing and a blood collection vacuum tube containing a lysis/binding buffer. Rest the patient&#39;s arm in a downward position and position the collection tube lower than the butterfly needle. Insert the butterfly needle into the vein of the patient and attach the blood collection vacuum tube to the small-bore extension. 
	NOTE: This will prevent back-flow.</li>
	<li>After the blood collection, mix the contents of the tube by flipping the tube 5 - 10 times. 
	NOTE: Due to the remaining vacuum in the blood collection tube containing 1.6 mL of lysis/binding buffer, the volume of the sample collected will automatically be 1.6 mL.</li>
	<li>Disinfect the outside of the tube using 70% ethanol.</li>
	<li>Incubate the tubes for 20 min at room temperature. 
	NOTE: The protocol can be paused here and the full blood collection tubes can be stored at -20 &#176;C, 5 &#176;C, 25 &#176;C or 37 &#176;C for at least 1 month.</li>
	<li>Continue directly to the simplified NA extraction method.</li>
</ol>
2. Simplified NA Extraction of the Whole Blood
<ol>
	<li>To purify NA from the lysis/binding buffer-inactivated collected blood, mix the contents of the tube by flipping the vacuum tube by hand 5 - 10x.</li>
	<li>Remove the lid of the tube carefully and discharge the lid.</li>
	<li>Pour the prepared aliquot of MGPs (1 mL) directly into the blood collection tube.</li>
	<li>Place a new lid from an unused blood collection tube on the tube containing the sample.</li>
	<li>Place a finger on the lid to ensure that the tube is tightly closed and mix the contents of the tube by flipping the blood collection tube by hand 5 - 10 times.</li>
	<li>Place the tube in the magnetic holder and keep a finger on the lid to ensure the tube is tightly closed.</li>
	<li>Flip the magnetic holder with the tube a few times by hand to make sure that all the MGPs are collected at the side of the tube with the magnet. 
	NOTE: The magnetic holder can be replaced with an elongated magnet and a rubber band.</li>
	<li>Remove the lid of the tube and discard the contents of the tube either by using a disposable pipette or simply by pouring the contents into a 50 mL collection tube. 
	NOTE: Avoid aerosols from the 50 mL collection tube by closing the tube with a lid.</li>
	<li>Pour the prepared aliquot of wash buffer-1 (WB-1, 4 mL) directly into the blood collection tube.</li>
	<li>Place the lid on the tube and place a finger on the lid to ensure the tube is tightly closed.</li>
	<li>Remove the tube from the magnetic holder, keeping the lid securely tightened with a finger. 
	NOTE: If using a magnet and a rubber band, simply remove the rubber band and magnet from the tube.</li>
	<li>Resuspend the MGPs by flipping the blood collection tube by hand 5 - 10 times.</li>
	<li>Repeat steps 2.6 - 2.8.</li>
	<li>Pour the prepared aliquot of WB-2 (1.5 mL) directly into the blood collection tube.</li>
	<li>Place the lid on the tube and remove the tube from the magnetic holder. 
	NOTE: If using a magnet and a rubber band, simply remove the rubber band and magnet from the tube.</li>
	<li>Place a finger on the lid to ensure the tube is tightly closed.</li>
	<li>Resuspend the MGPs by flipping the blood collection tube for a few seconds by hand.</li>
	<li>Repeat steps 2.6 - 2.8.</li>
	<li>Pour the prepared aliquot of WB-3 (3 mL) directly into the blood collection tube.</li>
	<li>Place the lid on the tube and remove the tube from the magnetic holder. 
	NOTE: If using a magnet and a rubber band, simply remove the rubber band and magnet from the tube.</li>
	<li>Place a finger on the lid to ensure the tube is tightly closed.</li>
	<li>Resuspend the MGPs by flipping the blood collection tube by hand 5 - 10 times.</li>
	<li>Repeat steps 2.6 - 2.8.</li>
	<li>Pour the prepared aliquot of elution buffer (EB, 100 &#181;L) directly into the blood collection tube.</li>
	<li>Place the lid on the tube and remove the tube from the magnetic holder. 
	NOTE: If using a magnet and a rubber band, simply remove the rubber band and magnet from the tube.</li>
	<li>Resuspend the MGPs in the EB by tapping the blood collection tube 5 - 10 times with a finger. 
	NOTE: The protocol can be paused here, and the tubes can be stored at -20 &#176;C.</li>
	<li>Transfer one droplet (5 - 8 &#181;L) of the resuspended MGPs to the downstream NA amplification reaction mix using a 1.5 mL disposable pipette (any downstream diagnostic NA amplification assay such as loop-mediated isothermal amplification (LAMP)/ Reverse-transcription LAMP (RT-LAMP) or quantitative polymerase chain reaction (qPCR)/RT-qPCR assays can be used). 
	NOTE: The NAs will stick to the MGPs, so be sure to use the MGPs in the downstream NA amplification reaction. Mix the MGP suspension before use. After mixing, the MGPs will collect at the bottom of the tube.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vacutainer K2 EDTA tubes (4 mL)</td>
			<td>Becton Dickinson</td>
			<td>368861</td>
			<td></td>
		</tr>
		<tr>
			<td>Plus Blood Collection</td>
			<td>Becton Dickinson</td>
			<td>362725</td>
			<td></td>
		</tr>
		<tr>
			<td>23G x 1 needle</td>
			<td>Becton Dickinson</td>
			<td>300800</td>
			<td></td>
		</tr>
		<tr>
			<td>LUER LOK syringe (3 mL)</td>
			<td>Becton Dickinson</td>
			<td>309658</td>
			<td></td>
		</tr>
		<tr>
			<td>Butterfly needle with small-bore extension tubing</td>
			<td>J&#248;rgen Kruuse A/S</td>
			<td>121714</td>
			<td></td>
		</tr>
		<tr>
			<td>1% Virkon</td>
			<td>Nomeco A/S</td>
			<td>849265</td>
			<td></td>
		</tr>
		<tr>
			<td>MagNA Pure LC DNA Isolation Kit I - Lysis/Binding Buffer - Refill</td>
			<td>Roche Diagnostics A/S</td>
			<td>3246752001</td>
			<td></td>
		</tr>
		<tr>
			<td>MagNA Pure LC Total Nucleic Acid Isolation Kit</td>
			<td>Roche Diagnostics A/S</td>
			<td>3038505001</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc&#8482; Biobanking and Cell Culture Cryogenic Tubes (1,8 mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>375418</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc&#8482; Biobanking and Cell Culture Cryogenic Tubes (3,6 mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>379189</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc&#8482; Biobanking and Cell Culture Cryogenic Tubes (4,5 mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>379146</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag&#8482;-5 Magnet</td>
			<td>Thermo Fisher Scientific</td>
			<td>12303D</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette (3.5 mL)</td>
			<td>Sarstedt</td>
			<td>86.1171.010</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific&#8482; Samco&#8482; Fine Tip Transfer Pipettes (1.5 mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>231</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific&#8482; Nunc&#8482; 50 mL Conical Sterile Polypropylene Centrifuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>339652</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Rosenstierne, M. W., et al. <a href="https://app.jove.com/v/58001">Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples.</a> J. Vis. Exp. (2018).
This video demonstrates a technique for nucleic acid extraction from whole human blood using magnetic glass particles (MGPs) &#8212; small magnetic beads coated with silica. Upon lysing the blood cells using a lysis buffer, the released nucleic acids bind to the surface of silica-coated MGPs, extracting the molecules from the sample.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines
1. Blood Collection from Patients
...

Take a tube containing freshly drawn human blood and add a lysis buffer.
The detergent in the buffer permeabilizes the cell membrane, lysing the cells and releasing intracellular nucleic acids, or NAs, and nucleases.
Chaotropic agents in the buffer denature nucleases, preventing NA degradation.
Add magnetic glass particles or MGPs &#160;&#8212; silica-coated magnetic particles &#8212; and mix thoroughly.
The buffer&#39;s low pH protonates silica, rendering a positive charge on the MGPs&#39; surface.
Negatively charged NAs bind electrostatically to the charged surface, forming an NA-MGP complex.
Under a magnetic field, NA-MGP complexes collect at the side of the tube.
Discard the supernatant containing cell debris and proteins.
Perform multiple washing steps by resuspending the NA-MGP complexes in a wash buffer, applying a magnetic field to collect the complexes, and discarding the supernatant with the remaining contaminants.
Resuspend complexes in an elution buffer. The high pH disrupts NA-MGP interaction, releasing the NAs. The eluted NAs are ready for downstream assays.

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Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

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A Technique for Magnetic Glass Particle-Mediated Nucleic Acid Extraction from Blood

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A Technique for Magnetic Glass Particle-Mediated Nucleic Acid Extraction from Blood