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,<....

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 ....

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