The authors have no acknowledgements.

1. Swab Sampling in the Field
<ol>
	<li>Wear a clean pair of gloves.</li>
	<li>Measure the size of the sampling area without touching the surface using a measuring tape or ruler. Try to estimate the area as accurately as possible and fill out a report form (Supplementary Table 1).</li>
	<li>Check the swab kit for possible leakages and label sample transport bags and swab kits.</li>
	<li>Move the swab across the sampling area as follows: one stroke in horizontal direction, one stroke in vertical direction, and one stroke in a diagonal direction. Do not swab a surface area larger than 700 cm2.</li>
	<li>Place each swab into a tube and tighten the cap securely.</li>
</ol>
2. Storage and Transport of Swabs to the Laboratory
<ol>
	<li>Keep swabs at 4 &#176;C for up to 48 h. If storage for longer periods is required, store the swabs at -20 &#176;C (or -70 &#176;C).</li>
	<li>Keep the tubes at 0-4 &#176;C (i.e., use cold packs) in an insulated container during transport to the laboratory and ship within 24-48 h of collection.</li>
</ol>
3. Virus Concentration, Viral RNA Extraction and Purification
NOTE: All centrifugation steps use a table top centrifuge at 5,000 x g for 5 min at room temperature, unless stated otherwise. Be extra careful when working with the universal nucleic acid extraction (UNEX) buffer. Wear goggles or face shield.
<ol>
	<li>Label one 15 mL tube and one RNA Midi column for each sample. Include a negative extraction control in each experiment.</li>
	<li>To prepare the Lysis solution for 10 swabs, mix 25 ml of UNEX buffer with 25 mL of PBST (PBS pH 7.2 containing 0.02% Tween-80).</li>
	<li>Add 50 &#181;L of coliphage MS2 suspension (106 PFU/&#181;l) to 50 mL of Lysis solution.</li>
	<li>Place a swab in a 15 mL tube and add 5 ml Lysis solution. Mix and incubate for 10 min at room temperature.</li>
	<li>Add 5 ml 100% ethanol to each tube and vortex for 10 s.</li>
	<li>Carefully remove the swab from the 15 mL tube (it contains UNEX buffer) pressing it gently against the side of the tube to remove excess liquid and then discard the swab. The remaining volumes should be between 9-10 mL.</li>
</ol>
4. Midi Column Viral Nucleic Acid Extraction
<ol>
	<li>Carefully transfer 4.5 mL UNEX/ethanol mixture from step 3.6 onto a midi column, centrifuge and discard the filtrate.</li>
	<li>Load another 4.5 mL from the same mixture onto the same column, centrifuge, and discard the filtrate.</li>
	<li>For the first wash, add 3.5 ml of 70% ethanol onto the Midi column, centrifuge and discard the filtrate.</li>
	<li>For the second wash, add another 3.5 ml of 70% ethanol onto the Midi column. Centrifuge and discard the filtrate.</li>
	<li>For the dry-spin, centrifuge the Midi columns to remove all traces of ethanol (which can negatively affect your viral RNA recovery).</li>
	<li>Place the Midi column in a new 15 ml centrifuge tube. Add 250 &#181;l elution buffer onto the Midi column; wait for 1 min before centrifuging to obtain the highest viral RNA recovery.</li>
	<li>Store the extracted nucleic acid at -70 &#176;C or proceed immediately to step 5.</li>
</ol>
5. Concentration of Viral Nucleic Acid Using RNA Clean and Concentrator Kits
<ol>
	<li>Add 500 &#181;L of RNA Binding Buffer to 250 &#181;L of RNA eluted in step 4.7 above and vortex for 10 s.</li>
	<li>Add 750 &#181;L 100% ethanol and vortex for 10 s.</li>
	<li>Label a spin column for each sample. Load the 750 &#181;L sample onto a spin column.</li>
	<li>Centrifuge the spin columns at 12,000 x g for 1 min. Discard the flow-through.</li>
	<li>Load the remaining 750 &#181;L onto a spin column and centrifuge at 12,000 x g for 1 min.</li>
	<li>For the pre-wash, add 400 &#181;L RNA Prep buffer to each spin column and centrifuge at 12,000 x g for 1 min. Discard the flow-through.</li>
	<li>For the first wash, add 800 &#181;L RNA Wash Buffer to each spin column and centrifuge at 12,000 x g for 1 min. Discard the flow-through.</li>
	<li>For the second wash, add 400 &#181;L RNA Wash Buffer to spin column and centrifuge at 12,000 x g for 1 min. Discard the flow-through.</li>
	<li>For the dry-spin, centrifuge the spin column to remove all traces of wash buffer at 12,000 x g for 2 min.</li>
	<li>Carefully transfer spin column to a clean 1.5 mL microcentrifuge tube.</li>
	<li>Add 25 &#181;L elution buffer onto the spin column and incubate for 1 min at room temperature. This will increase the recovery of viral RNA from the column.</li>
	<li>Collect RNA by centrifuging the spin column at 10,000 x g for 1 min.</li>
	<li>Either proceed directly to RT-qPCR (step 6) or store RNA at -70 &#176;C.</li>
</ol>
6. Mutiplex RT-qPCR Detection of Genogroup I, and II Noroviruses, and Coliphage MS 2 (Supplementary Table 2)
<ol>
	<li>Clean working surfaces, pipettes, and centrifuges with RNase decontamination solution to reduce possible contamination.</li>
	<li>Thaw RT-qPCR reagents on ice. Thaw RNA on ice (in a separate area/room).</li>
	<li>Determine the number of reactions and add at least 10% more of them (e.g. if 10 reactions are needed, make master mix for 11).</li>
	<li>Vortex individual master mix components, except for 25x RT-qPCR enzyme, for 5 s. Carefully mix the enzyme by flicking the tube with a finger.</li>
	<li>Briefly centrifuge master mix components for 5 s.</li>
	<li>Prepare the master mix for the realtime RT-PCR detection of norovirus GI and GII according to the kit instructions and add norovirus-specific oligonucleotide primers and probes in a 1.5 ml microcentrifuge tube (Supplementary Table 3).</li>
	<li>Mix the master mix by pipetting 5-10 times up and down (vortexing is not recommended). Aliquot 22 &#181;L of the master mix in each well of real time PCR 96-well plate.</li>
	<li>Vortex sample RNA for 5 sec, and collect by brief (5 s) centrifugation.</li>
	<li>Add 3 &#181;L of sample RNA and GI and GII positive controls to the RT-qPCR plate (follow template from Table 2). Add 3 &#181;L of nuclease-freewater in the no-template-control (NTC)wells.</li>
	<li>Seal the real-time plate with optical adhesive film.</li>
	<li>Carefully centrifuge the real-time plate at 1,300 x g for 1 min to remove any air bubbles or liquid drops that may be present in the wells.</li>
	<li>Set up a real-time PCR instrument and set-up the following thermocycling conditions: 1) RT step for 10 min. at 45 &#176;C (2) activation of Taq polymerase, 10 min. at 95 &#176;C and (3) 45 cycles of 15 s at 95 &#176;C, and 60 s at 60 &#176;C.</li>
</ol>
7. Quantification of Norovirus in Swab Samples
<ol>
	<li>Check the results of positive and negative controls to validate RT-qPCR results (Supplementary Table 3).</li>
	<li>Determine whether each standard curve meets the acceptable values given in Supplementary Table 2 and calculate the overall standard deviation for the standard curve using Equation 1. 
	(Equation 1) % efficiency = 100 x 10(average Ct Values-Intercept)/Slope.</li>
	<li>If the PCR thermal cycler software does not calculate the slope for each standard curve, determine slope and R2values by regression using statistic software (e.g. Excel, SPSS, or SAS). In addition, calculate the % efficiency for standard curves following equation I</li>
	<li>Record the RNA copy number calculated by the thermal cycler software for all test samples based upon standard curves that meet the criteria specified in Supplementary Table 4. Rerun any samples with standard curves that do not meet the criteria or have false-positive controls. Check Ct values of coliphage MS2, which was added as an internal control to monitor PCR inhibition.</li>
	<li>Determine the total number of RNA copies per sample by the multiplying the RNA copy number calculated in step 7.2 by the ratio of the volume (25 to 50 &#181;L) of total RNA eluent to that of the RNA (3 to 5 &#181;L) used for the RT-qPCR reaction. Alternatively, calculate the RNA density by dividing the total RNA copy number by the object surface area (cm2) analyzed.</li>
</ol>
8. Genotyping of Real-time RT-PCR Positive Samples by Hemi Nested Conventional PCR Amplification
<ol>
	<li>Prepare the first round of master mix for each primer set of the RT-PCR assay (Supplementary Tables 2 , and 5).</li>
	<li>Add 5 &#181;L of norovirus positive RNA to 20 &#181;L of master mix.</li>
	<li>Run the RT-PCR under the following conditions: (1) RT step for 30 min at 42 &#176;C (2) activation of Taq polymerase, 15 min at 95 &#176;C, and (3) 40 cycles of 30 s at 95 &#176;C, 30 s at 50 &#176;C, and 60 sec at 72 &#176;C. After 40 cycles, incubate for an additional 10 min at 72 &#176;C.</li>
	<li>Prepare the second round of mater mix for each primer set of the RT-PCR assay (Supplementary Tables 2 and 5).</li>
	<li>Add 23 &#181;L master mix and add 2 &#181;L of first-round RT-PCR products (from first round) to each tube (ideally the first-round product should be diluted 1/10 in RNase-free water).</li>
	<li>Repeat step 8.3.</li>
	<li>Prepare a 2% agarose gel in 100 ml 1x Tris-acetate EDTA (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA, at pH 8.3). After dissolving agarose by heating the mixture in a microwave oven, add 10 &#181;L of nucleic acid stain per 100 mL of prepared gel after cooling the mixture to 60-70 &#176;C, pour the gel and insert the combs. Let the gel settle for at least 30 min.</li>
	<li>Mix 15 &#181;l of each RT-PCR product with 3 &#181;L of 6x loading dye. Load 15 &#181;L of each sample and electrophorese the agarose gel at 100 V for 1 h.</li>
	<li>Excise target RT-PCR fragments of appropriate size (330 bp for GI and 341 bp for GII) from the gel and purify RNA using a commercial gel extraction kit. The purified PCR product can now be used for Sanger sequencing.</li>
</ol>

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Authors have no conflicting interest. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

Noroviruses have a 50% human infectious dose between 18 and 103 virus particles20. Therefore, even low-level contamination of surfaces may pose a public health risk. Several aspects of the swab sampling protocol were evaluated including: 1) different swab materials, 2) storage condition swabs during transport, 3) viral RNA concentration, and 4) coliphage MS2 as internal extraction control.
Until recently, only the performance of swabs made from cotton, polyes...

Human noroviruses are a leading cause of epidemic and sporadic acute gastroenteritis worldwide 1,2,3. The virus is extremely contagious and transmission occurs through direct person to person interaction or indirectly through contact with contaminated food, water or environmental surfaces. Noroviruses can be shed for extended periods and prolonged survival of the virus on environmental surfaces has been documented 1,2,3. During peak shedding, billions of virus particles are released per gram of feces, and vomit also contains a sufficient number of viral particles to cause infection 4,5,6,7,8,9,10. In addition, transfer of the virus between inanimate surfaces and human skin can occur easily 2,11,12. Hence, monitoring of environmental contamination may assist in outbreak investigations and in assessing the effectiveness of clean-up and disinfection procedures.
Several environmental sampling protocols have been described for the detection of rotavirus, coliphage MS2, feline calicivirus (FCV), and bacteriophage P22 13,14,15,16. However, the validation conditions described in&#160;these studies, including fast desiccation (&#60;1 hr) and small surface areas (25 x 100 cm2), may not adequately represent field settings. In addition, the expected low contamination levels of environmental surfaces require protocols that are able to detect very few virus particles.
We developed a macrofoam-based surface sampling method for the detection and typing of norovirus. This method has been validated during several norovirus outbreaks. The protocol includes 1) how to collect swab samples from environmental surfaces (2) how to best maintain integrity of the samples during collection and shipping to the laboratory, and 3) laboratory testing and typing of norovirus.

<table border="0" cellpadding="0" cellspacing="0" width="1094">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Generic name for kits</td>
			<td style="width:259px;"></td>
			<td style="width:153px;"></td>
			<td style="width:185px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Macrofoam swab</td>
			<td>Premoistened EnviroMax Swab kit&#160;</td>
			<td>Puritan</td>
			<td>2588060PFUW</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;RNA Lysis buffer&#160;</td>
			<td>CDC UNEX buffer</td>
			<td>Microbiologics</td>
			<td>Cat No MR0501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNA extraction spin column</td>
			<td>Midi column</td>
			<td>Omega Biotek</td>
			<td>Cat No R6664-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">RNA purification spin column</td>
			<td>Zymol RNA Clean and Concentrator kit&#160;</td>
			<td>Zymo Research</td>
			<td>Cat No R1016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Real time RT-PCR kit</td>
			<td>AgPath kit One-Step RT-PCR Kit</td>
			<td>Life Technologies</td>
			<td>Cat No 4387391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conventional RT-PCR kit</td>
			<td>Qiagen one step RT-PCR kit</td>
			<td>Qiagen kit</td>
			<td>Cat No 210212</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Gel extraction kit</td>
			<td>Qiagen QIAquick gel extraction kit</td>
			<td>Qiagen kit</td>
			<td>Cat No 28704 or 28706</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;"></td>
			<td>Coliphage MS2</td>
			<td>ATCC</td>
			<td>Cat No 15597-B1</td>
			<td></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;"></td>
			<td>RNA run-off transcripts</td>
			<td></td>
			<td></td>
			<td style="width:285px;">Bacteriophage MS2 (ATCC No. 15597-B1) can be cultivated using Escherichia coli (E.coli) Famp (ATCC No. 700891).&#160;</td>
		</tr>
		<tr height="148">
			<td height="148" style="height:148px;"></td>
			<td>Realtime PCR platform</td>
			<td>Applied Biosystems</td>
			<td>Model ABI 7500</td>
			<td style="width:285px;">GI and GII RNA run off transcripts were quantified spectrophotometrically at A260, diluted in diethyl pyrocarbonate-treated water to 1 &#215; 106 copies/ &#956;l, and stored at &#8722;80&#176;C with 1.0 U /&#956;l RNasin (Promega, Madison, WI).&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td>Optical 96-well reaction plate</td>
			<td>Thermo Scientific</td>
			<td>Cat No 4316813</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td><a href="http://www.thermofisher.com/order/catalog/en/US/adirect/%20https:/www.thermofisher.com/order/catalog/en/US/adirect/lt?cmd=catProductDetail&amp;showAddButton=true&amp;productID=4306311&amp;_bcs_=H4sIAAAAAAAAAI1RTWvDMAz9Nb4srDg2yXpNW3rZVyHbuRhHSQx2HWwnJf9%2BUkO6scMYGEmWnqX3%0A5Mec8e0p%2BGbUKWZMlFkNYTIa4h%2F5PqWByYqJI57r9bpJPQTnWxPRb7R3mB4jGrig6b0DdD40WOyT%0As9iBCUmHb1MYge78iYi06ZznPEd0exacF6LkD5h%2BNTr4yg1II0DH5C6rmh6imSA7GuuInsIq5TUy%0AjD6Yf%2FKU1Zav3NBrlZT13cr8s0ajGhNAJ4xsYvKoXcPkAYEHEwer5jrNFuWVdF1ieajNpbPwAhPY%0AvUrQ%2BTAjAt88w4zlm8Ly%2FYRha2yiyeUSUJM35ajHt%2BZiUS0KuWNityrHkLSjuyHUDbkgfuxgbfwx%0AD9Q0vydo9rrs4te675%2FTKhvpd74AhB0C9yUCAAA%3D&amp;returnURL=http%3A%2F%2Fwww.thermofisher.com%3A80%2Forder%2Fcatalog%2Fen%2FUS%2Fadirect%2Flt%3Fcmd%3DcatDisplayStyle%26catKey%3D101%26filterType%3D1%26OP%3Dfilter%26filter%3Dft_1101%252Ff_2005260*">MicroAmp Clear Adhesive Film&#160;</a></td>
			<td>Thermo Scientific</td>
			<td>Cat No 4306311</td>
			<td></td>
		</tr>
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</table>

swab sampling method for the detection of human norovirus on surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

Figure 1 presents a flowchart of the swab sampling protocol. This protocol consists of four main steps; 1) sample collection, 2) sample storage and transportation, 3) viral RNA purification and concentration and 4) RT-qPCR assay and genotyping.
<img alt="Figure 1" src="/files/ftp_upload/55205/55205fig1.jpg" /> 
Figure 1: Flow chart of the final protocol for environmental surface sampling of noroviru...

Watch this Scientific Journal Video about Swab Sampling Method for the Detection of Human Norovirus on Surfaces at JoVE.com

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

swab-sampling-method-for-the-detection-of-human-norovirus-on-surfaces

Research

JoVE Journal

Immunology and Infection

19.5K Views.  Centers for Disease Control and Prevention. A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces. 

Video: Swab Sampling Method for the Detection of Human Norovirus on Surfaces

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 ...

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

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, ...

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

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 ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...