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

Summary

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).

Abstract

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.

Introduction

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 sample^2,3,4,5, were used during the outbreak; however, these methods require 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.

Protocol

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 irritants. Take appropriate laboratory safety measures, use a flow hood, and wear gloves when handling it. Avoid any skin and eye contact. If the buffer is spilled, the contaminated surface must never be disinfected directly with chloramine or sodium hypochlorite (the active ingredients in "bleach") because this mixture may lead to the formation of toxic cyanide. First, wipe up the spilled buffer with the absorbent tissue. Next, clean the surface with 70% ethanol and then with water, and finally, use chloramine or sodium hypochlorite.

1. To prepare the blood collection vacuum tubes, inject 1.6 mL of the specific commercial lysis buffer into a 4 mL EDTA vacuum tube by puncturing the lid of the tube using a 25 G x 1 needle and a 3 mL syringe.
   NOTE: Do not remove the lid of the vacuum tube. The vacuum must be maintained.
2. Store the vacuum tubes containing the buffer at room temperature until use.
   NOTE: The tubes are stable for at least 1 year after their preparation.

2. Preparation of Buffers for NA Extraction

1. To prepare the buffers for an NA extraction, place two 1.8 mL, two 4.5 mL, and one 3.6 mL tubes in a rack.
2. Add, by pipetting, 960 µL of magnetic glass particles (MGPs) to a clean 1.8 mL tube and label it with MGPs. Resuspend the MGP suspension completely before pipetting it.
   NOTE: The MGPs tend to quickly collect at the bottom of the tube.
3. Add, by pipetting, 4 mL of wash buffer I to a clean 4.5 mL tube and label it as WB-1.
   CAUTION: Wash buffer I contains guanidinium chloride, which is irritant. Take appropriate laboratory safety measures, use a flow hood, and wear gloves when handling it. Avoid any skin and eye contact.
4. Add, by pipetting, 1.5 mL of wash buffer II to a clean 3.6 mL tube and label it as WB-2.
5. Add, by pipetting, 3 mL of wash buffer III to a clean 4.5 mL tube and label it as WB-3.
6. Add, by pipetting, 100 µL of elution buffer to a clean 1.8 mL tube and label it as EB.
7. Store the aliquoted buffers at room temperature until use.
   NOTE: The tubes are stable for at least 1 month after their preparation.

3. Blood Collection from Patients with Signs and Symptoms of a Virus Infection

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.

1. 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'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.
2. 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.
3. Disinfect the outside of the tube using 70% ethanol.
4. 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 °C, 5 °C, 25 °C or 37 °C for at least 1 month⁷.
5. Continue directly to the simplified NA extraction method.

4. Simplified NA Extraction of the Whole Blood

1. 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.
2. Remove the lid of the tube carefully and discharge the lid.
3. Pour the prepared aliquot of MGPs (1 mL) directly into the blood collection tube.
4. Place a new lid from an unused blood collection tube on the tube containing the sample.
5. 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.
6. Place the tube in the magnetic holder and keep a finger on the lid to ensure the tube is tightly closed.
7. 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.
8. 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.
9. Pour the prepared aliquot of WB-1 (4 mL) directly into the blood collection tube.
10. Place the lid on the tube and place a finger on the lid to ensure the tube is tightly closed.
11. 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.
12. Resuspend the MGPs by flipping the blood collection tube by hand 5 - 10 times.
13. Repeat steps 4.6 - 4.8.
14. Pour the prepared aliquot of WB-2 (1.5 mL) directly into the blood collection tube.
15. 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.
16. Place a finger on the lid to ensure the tube is tightly closed.
17. Resuspend the MGPs by flipping the blood collection tube for a few seconds by hand.
18. Repeat step 4.6 - 4.8.
19. Pour the prepared aliquot of WB-3 (3 mL) directly into the blood collection tube.
20. 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.
21. Place a finger on the lid to ensure the tube is tightly closed.
22. Resuspend the MGPs by flipping the blood collection tube by hand 5 - 10 times.
23. Repeat steps 4.6 - 4.8.
24. Pour the prepared aliquot of EB (100 µL) directly into the blood collection tube.
25. 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.
26. 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 °C.
27. Transfer one droplet (5 - 8 µL) 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 LAMP/RT-LAMP or 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.

Results

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 (

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Vacutainer K2 EDTA tubes (4 mL)	Becton Dickinson	368861
Plus Blood Collection	Becton Dickinson	362725
23G x 1 needle	Becton Dickinson	300800
LUER LOK syringe (3 mL)	Becton Dickinson	309658
Butterfly needle with small-bore extension tubing	Jørgen Kruuse A/S	121714
1% Virkon	Nomeco A/S	849265
MagNA Pure LC DNA Isolation Kit I - Lysis/Binding Buffer - Refill	Roche Diagnostics A/S	03246752001
MagNA Pure LC Total Nucleic Acid Isolation Kit	Roche Diagnostics A/S	3038505001
Nunc™ Biobanking and Cell Culture Cryogenic Tubes (1,8 mL)	Thermo Fisher Scientific	375418
Nunc™ Biobanking and Cell Culture Cryogenic Tubes (3,6 mL)	Thermo Fisher Scientific	379189
Nunc™ Biobanking and Cell Culture Cryogenic Tubes (4,5 mL)	Thermo Fisher Scientific	379146
DynaMag™-5 Magnet	Thermo Fisher Scientific	12303D
Transfer pipette (3.5 mL)	Sarstedt	86.1171.010
Thermo Scientific™ Samco™ Fine Tip Transfer Pipettes (1.5 mL)	Thermo Fisher Scientific	231
Thermo Scientific™ Nunc™ 50 mL Conical Sterile Polypropylene Centrifuge Tubes	Thermo Fisher Scientific	339652