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

We thank Susanne Lopes Rasmussen and Solvej Kolbj&#248;rn Jensen for their technical assistance and for handling the clinical samples. This project is part of the EbolaMoDRAD consortium, which has received funding from the Innovative Medicine Initiative 2 Joint Undertaking under grant agreement N&#176;115843. This Joint Undertaking receives support from the European Union's Horizon 2020 research and innovation program and EFPIA.

The Committee on Biomedical Research Ethics, Capital region has given informed consent, and all methods described here have been exempted from a review by the ethical committee system, in accordance with the Danish law on assay development projects.
1. Preparation of Blood Collection Vacuum Tubes for Virus Inactivation and Rapid NA Extraction
CAUTION: The buffer used for this protocol contains guanidinium thiocyanate (GITC) and a non-ionic surfactant, which are irrita...

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In this report, we describe a safe, rapid, and simple manual bedside NA extraction method for the downstream molecular detection of a virus in lysis/binding buffer-inactivated whole blood. The described NA extraction method was developed to be performed directly on whole blood samples collected in vacuum blood collection tubes containing the lysis/binding buffer (Table of Materials)7. This specific buffer inactivates EBOV7 and is the critical component in t...

In virus outbreak situations, when patients are confined to the hospital isolation wards or when a fast diagnosis is needed, a safe, simple, and accurate point-of-care molecular diagnosis is imperative for the patient care and risk containment. The two recent viral outbreaks, of the Ebola virus (EBOV) in West Africa (2013) and the Zika virus in South America (2015), have increased the interest in improved point-of-care molecular diagnostic tests, such as reverse transcription loop-mediated isothermal amplification (RT-LAMP)1,2 and recombinase polymerase amplification (RPA)3,4. Both RT-LAMP and RPA are rapid, sensitive, and specific molecular tests that can be performed on simplified sample preparations. For the Zika virus, RT-LAMP has been combined with a lateral flow assay (LFA), which can detect Zika virus in non-purified whole blood samples within 30 min1; however, for EBOV, which is classified as a risk group 4 pathogen and is highly contagious, the samples need to be handled under biosafety level 4 (BSL-4) conditions and inactivated before any safe diagnostic procedures can be performed.
Simplified inactivation methods for EBOV, such as the addition of lysis buffers to the sample2,3,4,5, were used during the outbreak; however, these methods require&#160;handling under BSL-4 conditions with laboratory equipment, such as BSL-3 biosafety cabinets, centrifuges, heating blocks, and pipettes, at a minimum. This equipment is normally not present in isolation wards or out in field hospitals. To overcome this challenge, attempts have been made to perform diagnostics in suitcases3, and several portable devices and machines have been developed [e.g., a portable device for nucleic acid (NA) extraction]6. However, EBOV-positive samples still need to be inactivated before these devices can be used.
We have previously reported a rapid bedside virus inactivation method for the EBOV7, Vaccinia virus, and Cowpox virus8 by addition of a commercial lysis/binding buffer to ordinary vacuum blood collection tubes, allowing for the direct transfer of blood from the patient into the inactivation buffer7. This direct and immediate inactivation in a closed system eliminates the need for handling the samples using any rigorous containment, such as BSL-4 conditions7, and the samples can be handled under normal BSL-2 conditions. This inactivation method is compatible with several NA extraction systems, such as robots and hand purification kits7; however, these methods require laboratory equipment, such as robots, centrifuges, and electricity, which are not always present in field settings or inside hospital isolation wards.
In this report, we describe a safe, rapid, and simplified manual NA extraction method for the subsequent molecular detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction method does not require any equipment other than a magnet/magnetic holder. No centrifuges, heating blocks, or electricity are needed for the NA extraction. Hence, this method is not dependent on laboratory facilities and can easily be used anywhere (e.g., in field hospitals, in hospital isolation wards, or with low-resource settings). The NA extraction method is rapid and simple and can be used directly in any downstream NA tests, such as qPCR, RT-qPCR, LAMP, or RT-LAMP. When this NA extraction method is combined with LAMP and a portable battery-driven isothermal instrument, a bedside diagnosis can be obtained within 40 min of the blood collection.

<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

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

The protocol presented here is simple and efficient and can be broadly applied to any molecular assay to be performed on infectious whole blood samples inactivated with the lysis/binding buffer. The workflow for the blood inactivation and NA extraction is shown in Figure 1, including the preparation of blood collection vacuum tubes7 (Figure 1A), the blood collection7 (<strong class=...

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

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

This method can help when performing point of care diagnostics, of patients with signs of viral infections. The main advantage of this technique is that it can be performed without laboratory equipment and electricity, and hence it can be performed in a hospital ward. Demonstrating the blood collection procedure, will be Medical Doctor Anders Fomsgaard, and Scientist Morten Rasmussen, both from my laboratory.Begin by preparing the blood collection tubes. Use a 25 gauge one inch needle attached to a 3ML syringe, to inject 1.6ML of a commercial lysis buffer into a 4ML EDTA vacuum tube. It is critical to maintain the vacuum in the blood collection tube for blood collection.Hence, always use a needle to inject the lysis buffer into the blood collection tube. Next completely re-suspend a 960 micro-liter volume of magnetic glass particles, or MGPs, and transfer to a labeled 1.8ML tube. Next pipet 4ML of wash buffer one into a 4.5ML tube labeled WB-1.1.5ML of wash buffer two into a 3.6ML tube labeled WB-2, and 3ML of wash buffer three into a 4.5ML tube labeled WB-3.Finally add 100 micro-liters of elution buffer to a 1.8ML tube labeled EB, and leave the tubes at room temperature until use. To collect intravenous whole blood from the patient, use a butterfly needle with a small bore extension tubing, and one of the previously prepared blood collection vacuum tubes containing lysis buffer. Rest the patient's arm in a downward position.Insert the butterfly needle into the vein of the patient, and attach the blood collection vacuum tube to the small bore extension. After blood collection mix the contents of the tube, by flipping the tube five to ten times. Disinfect the outside of the tube using 70%ethanol.Then incubate the tubes for 20 minutes at room temperature for virus inactivation. To purify nucleic acids from the collected blood, again mix the contents of the tube by flipping the vacuum tube by hand five to ten times. Pour the prepared aliquot of MGP's directly into the blood collection tube.The 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 five to ten 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 MGP's 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 50ML collection tube, using a disposable pipet. Pour the prepared aliquot of WB-1 directly into the blood collection tube and close the lid on the tube.Mix the content by hand five to ten times. Then collect the beads at the side of the tube, using the magnet and remove the liquid as before. Re-suspend the beads in the prepared aliquot of WB-2, then discard the solution, and repeat the procedure with a prepared aliquot of WB-3.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 re-suspend the MGP's by tapping five to ten times with a finger. Finally, use a 1.5ML disposable pipet to transfer one droplet of the re-suspended MGP's to the downstream nucleic acid amplification reaction mix.Be sure to fully re-suspend the MGP's before transferring only one droplet into the PCR reaction. To demonstrate proof of concept, negative whole blood was spiked with 10 fold dilutions of the Ebola virus standard, prepared from the recent outbreak in southern Guinea, or three different concentrations of Epstein Barr virus. The extracted nucleic acids were analyzed by in-house virus specific R2 qPCR or qPCR.The lower the crossing threshold, or CT, the higher the viral genome copy number. Here, whole bloods spiked with different concentrations of Ebola virus was detected by an Ebola virus specific lamp. The final concentration of virus in the spiked whole blood sample is seen on the X axis, and the minutes to positive is seen on the Y axis.Here, whole blood was spiked with 10 fold dilutions of a hepatitis C WHO standardized serum sample, with detection by HCV specific RT qPCR. Finally, this image shows the results of an Epstein Barr virus specific lamp reaction, on Epstein Barr virus positive whole blood. While attempting this procedure it's important to use the specified lysis binding buffer.This is a crucial step, and a crucial re-agent for the method. This lysis binding buffer will inactivate virus in the sample. Following this technique you can perform different molecular detection methods like lamp and PCR, and hence you can perform a differential diagnosis.

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