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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Immunology and Infection

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...