Name: rapid, safe, and simple manual bedside nucleic acid extraction for the detection of virus in whole blood samples
Uploaded: 2018-06-30T15:00:00.000+00:00
Duration: 6 min 59 s
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 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 &#34;bleach&#34;) 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.
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Preparation of Buffers for NA Extraction
<ol>
	<li>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.</li>
	<li>Add, by pipetting, 960 &#181;L 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.</li>
	<li>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.</li>
	<li>Add, by pipetting, 1.5 mL of wash buffer II to a clean 3.6 mL tube and label it as WB-2.</li>
	<li>Add, by pipetting, 3 mL of wash buffer III to a clean 4.5 mL tube and label it as WB-3.</li>
	<li>Add, by pipetting, 100 &#181;L of elution buffer to a clean 1.8 mL tube and label it as EB.</li>
	<li>Store the aliquoted buffers at room temperature until use. 
	NOTE: The tubes are stable for at least 1 month after their preparation.</li>
</ol>
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.
<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 month7.</li>
	<li>Continue directly to the simplified NA extraction method.</li>
</ol>
4. 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 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 4.6&#160;- 4.8.</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 step 4.6&#160;- 4.8.</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 4.6&#160;- 4.8.</li>
	<li>Pour the prepared aliquot of 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 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.</li>
</ol>

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J., Weller, 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=Buffer+AVL+alone+does+not+inactivate+Ebola+virus+in+a+representative+clinical+sample+type.">Buffer AVL alone does not inactivate Ebola virus in a representative clinical sample type.</a> Journal of Clinical Microbiology. 53 (10), 3148-3154 (2015).</li><li>Byrnes, S., Fan, 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=A+Portable,+Pressure+Driven,+Room+Temperature+Nucleic+Acid+Extraction+and+Storage+System+for+Point+of+Care+Molecular+Diagnostics.">A Portable, Pressure Driven, Room Temperature Nucleic Acid Extraction and Storage System for Point of Care Molecular Diagnostics.</a> Analytical Methods. 5 (13), 3177-3184 (2013).</li><li>Rosenstierne, M. 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The authors have nothing to disclose.

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><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&amp;oslash;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>03246752001</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&amp;trade; Biobanking and Cell Culture Cryogenic Tubes (1,8 mL)</td><td>Thermo Fisher Scientific</td><td>375418</td><td></td></tr><tr><td>Nunc&amp;trade; Biobanking and Cell Culture Cryogenic Tubes (3,6 mL)</td><td>Thermo Fisher Scientific</td><td>379189</td><td></td></tr><tr><td>Nunc&amp;trade; Biobanking and Cell Culture Cryogenic Tubes (4,5 mL)</td><td>Thermo Fisher Scientific</td><td>379146</td><td></td></tr><tr><td>DynaMag&amp;trade;-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&amp;trade; Samco&amp;trade; Fine Tip Transfer Pipettes (1.5 mL)</td><td>Thermo Fisher Scientific</td><td>231</td><td></td></tr><tr><td>Thermo Scientific&amp;trade; Nunc&amp;trade; 50 mL Conical Sterile Polypropylene Centrifuge Tubes</td><td>Thermo Fisher Scientific</td><td>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 ...

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Universidad de Cartagena UDEC

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Immunology and Infection

7.3K Views.  Statens Serum Institut. 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). 

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

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

Environment

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively).

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. Consequently, the sample preparation format is compatible with most liquid handling instruments, and can be used for many medium to high-throughput clinical applications and sample types.

TruTip is a simple nucleic acid extraction  technology whereby a porous, monolithic binding matrix is inserted into a  pipette tip. The geometry of the ...

Bioengineering

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Genetics

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from clinical and biological sources. The method is unbiased and universal; it uses random primers for cDNA synthesis and requires no prior knowledge of the viral sequence content. Before library construction, selective RNase H-based digestion is used to deplete unwanted RNA &#8212; including poly(rA) carrier and ribosomal RNA &#8212; from the viral RNA sample. Selective depletion improves both the data quality and the number of unique reads in viral RNA sequencing libraries. Moreover, a transposase-based &#39;tagmentation&#39; step is used in the protocol as it reduces overall library construction time. The protocol has enabled rapid deep sequencing of over 600 Lassa and Ebola virus samples-including collections from both blood and tissue isolates-and is broadly applicable to other microbial genomics studies.

This protocol describes a rapid and broadly applicable method for unbiased RNA-sequencing of viral samples from human clinical isolates.

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from ...

Medicine

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent.
At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes.
The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of use.

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food ...

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.

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 ...