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, polyester, nylon and antistatic wipe) had been evaluated and proposed for field use13,14,15,21,22. Of these, cotton swabs have been recommended by the ISO 15216 protocol for detection of norovirus and hepatitis A virus from food preparation surfaces and fomites23. However, there are limited data on the testing performance of cotton swabs under field conditions with large high-touch surfaces such as doorknobs, computers, and toilet seats that have been frequently implicated in the transmission of noroviruses24,25. Additionally, the size of these environmental surfaces exceeds the capacity of fiber-tipped swabs. In our study, we demonstrated that macrofoam swabs, which have a larger swab head than the fiber-tip swabs, have higher recovery rates for human norovirus when sampling environmental surfaces with an area up to 700 cm2.
Noroviruses in solution are relatively unstable at ambient temperature and require at least refrigeration. Therefore, shipping to the laboratory should occur at 0-4 &#176;C within 48 h after collection of the swabs. The volume of sample that can typically be tested in a PCR reaction is much smaller than that of&#160;the amount of sample after elution from the swab (3-5 &#956;L vs. 5-10 mL) which, without concentration, significantly reduces the positivity rate. In environmental virology, polyethylene glycol (PEG) has been used successfully to concentrate viruses from large volumes of environmental waters. However, &#160;volumes up to 10 mL can be easily extracted and concentrated using Midi spin columns to volumes as low as 250 &#956;L, and further concentrated 10-fold with high recovery using spin columns.
Although RT-qPCR is the gold standard for sensitive detection and quantification of norovirus, the sensitivity of these assays can be easily affected by the presence of PCR inhibitors that are co-extracted from the swab samples. Therefore, inclusion of an extraction control such as coliphage MS2 virus particles, in a multiplex PCR format that detects both norovirus as well as MS2, helps to monitor the presence of PCR inhibition and to assess variation among different experiments.
The test variables of a laboratory validation method are critical to determine the discriminatory power of a test method and are further translated into the method&#39;s likely field sampling performance. Taking real world field condition&#160;(e.g., delayed sampling, larger surface areas of up to 700 cm2) into account, the detection limit for norovirus was 3.4 log10(stainless steel) and 4 log10(toilet seat) copies norovirus per surface sampled6. Consequently, negative swab results are not definitive that no viral contamination has occured. Clinical and epidemiological information of norovirus illness always trumps environmental findings2,6.
Our data from a cruise ship showed that high-touch surfaces such as telephones, computer keyboards, and door handles tested positive for norovirus in cabins where people with reported viral gastroenteritis had stayed. These surfaces either became contaminated through direct contact or indirectly through droplets produced during toilet flushing or vomiting episodes6,7,8,9,10.
In conclusion, detection of norovirus on environmental surfaces using this newly developed swab protocol may assist in determining the level of environmental contamination during outbreaks, detect virus when clinical samples are not available and in monitoring of the effectiveness of cleaning practices. Fiber tipped swab (cotton and rayon) based surface sampling methods have been widely used to investigate other enteric viruses (e.g. rotavirus, and adenovirus)27,28,29. Considering the higher recovery efficiency for norovirus of macrofoam swabs over those fibers tipped swabs6, we expected that the macrofoam swabs will be able to detect other enteric viruses as well.

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><tbody><tr><td>&lt;strong&gt;Generic name for kits&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Macrofoam swab&lt;/strong&gt;</td><td>Premoistened EnviroMax Swab kit&amp;nbsp;</td><td>Puritan</td><td>2588060PFUW</td></tr><tr><td>&lt;strong&gt;RNA Lysis buffer&amp;nbsp;&lt;/strong&gt;</td><td>CDC UNEX buffer</td><td>Microbiologics</td><td>Cat No MR0501</td></tr><tr><td>&lt;strong&gt;RNA extraction spin column&lt;/strong&gt;</td><td>Midi column</td><td>Omega Biotek</td><td>Cat No R6664-02</td></tr><tr><td>&lt;strong&gt;RNA purification spin column&lt;/strong&gt;</td><td>Zymol RNA Clean and Concentrator kit&amp;nbsp;</td><td>Zymo Research</td><td>Cat No R1016</td></tr><tr><td>&lt;strong&gt;Real time RT-PCR kit&lt;/strong&gt;</td><td>AgPath kit One-Step RT-PCR Kit</td><td>Life Technologies</td><td>Cat No 4387391</td></tr><tr><td>&lt;strong&gt;Conventional RT-PCR kit&lt;/strong&gt;</td><td>Qiagen one step RT-PCR kit</td><td>Qiagen kit</td><td>Cat No 210212</td></tr><tr><td>&lt;strong&gt;Gel extraction kit&lt;/strong&gt;</td><td>Qiagen QIAquick gel extraction kit</td><td>Qiagen kit</td><td>Cat No 28704 or 28706</td></tr><tr><td></td><td>Coliphage MS2</td><td>ATCC</td><td>Cat No 15597-B1</td></tr><tr><td></td><td>RNA run-off transcripts</td><td></td><td></td></tr><tr><td></td><td>Realtime PCR platform</td><td>Applied Biosystems</td><td>Model ABI 7500</td></tr><tr><td></td><td>Optical 96-well reaction plate</td><td>Thermo Scientific</td><td>Cat No 4316813</td></tr><tr><td></td><td>&lt;a href=&quot;http://www.thermofisher.com/order/catalog/en/US/adirect/%20https:/www.thermofisher.com/order/catalog/en/US/adirect/lt?cmd=catProductDetail&amp;amp;showAddButton=true&amp;amp;productID=4306311&amp;amp;_bcs_=H4sIAAAAAAAAAI1RTWvDMAz9Nb4srDg2yXpNW3rZVyHbuRhHSQx2HWwnJf9%2BUkO6scMYGEmWnqX3%0A5Mec8e0p%2BGbUKWZMlFkNYTIa4h%2F5PqWByYqJI57r9bpJPQTnWxPRb7R3mB4jGrig6b0DdD40WOyT%0As9iBCUmHb1MYge78iYi06ZznPEd0exacF6LkD5h%2BNTr4yg1II0DH5C6rmh6imSA7GuuInsIq5TUy%0AjD6Yf%2FKU1Zav3NBrlZT13cr8s0ajGhNAJ4xsYvKoXcPkAYEHEwer5jrNFuWVdF1ieajNpbPwAhPY%0AvUrQ%2BTAjAt88w4zlm8Ly%2FYRha2yiyeUSUJM35ajHt%2BZiUS0KuWNityrHkLSjuyHUDbkgfuxgbfwx%0AD9Q0vydo9rrs4te675%2FTKhvpd74AhB0C9yUCAAA%3D&amp;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*&quot;&gt;MicroAmp Clear Adhesive Film&amp;nbsp;&lt;/a&gt;</td><td>Thermo Scientific</td><td>Cat No 4306311</td></tr></tbody></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 norovirus <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55205/55205fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1 summarizes the results from 34 swab samples that were collected from a cruise ship that had reported cases of suspected norovirus gastroenteritis during a voyage. Swab samples were extracted and tested in duplicate for norovirus by multiplex real-time RT-qPCR. Seventeen (18.5%) samples tested positive including 8 samples from surfaces in cabins where passengers with norovirus symptoms had stayed and 9 from common areas on the ship. The number of genomic copies per sample were calculated from the Ct values (which ranged from 16 to 31) using a standard curve of a norovirus GII.7 RNA transcript. The median number of genome&#160;copies from swab samples in cabins was 3.6 log10(range: 2.4 &#8211; 4.5 log10 RNA copies), which was significantly higher than the genome copies from common areas (range: 1.2 &#8211; 2.1 log10 genome copies) (P &#60;0.001). Four (23.5%) of the 17 positive swab samples were able to be genotyped, and all samples had identical GII.1 sequences.
<table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Location environmental swap collecteda</td>
			<td>Sample Point Description</td>
			<td>Average Ct value (# positive of the total number of samples tested)</td>
			<td>Genotype</td>
			<td>Norovirus RNA copy number per sampled areac</td>
		</tr>
		<tr>
			<td>Atrium</td>
			<td>Handrails</td>
			<td>34.3 (1/2)</td>
			<td>GII</td>
			<td>16</td>
		</tr>
		<tr>
			<td>Cabin A</td>
			<td>Toilet seat</td>
			<td>31.4 (2/2)</td>
			<td>GII.b</td>
			<td>31,217</td>
		</tr>
		<tr>
			<td>Cabin A</td>
			<td>Faucet</td>
			<td>37.5 (1/2)</td>
			<td>GII</td>
			<td>491</td>
		</tr>
		<tr>
			<td>Cabin A</td>
			<td>Door handle</td>
			<td>35.0 (2/2)</td>
			<td>GII</td>
			<td>2,675</td>
		</tr>
		<tr>
			<td>Cabin A</td>
			<td>Remote control</td>
			<td>38.6 (2/2)</td>
			<td>GII.1b</td>
			<td>233</td>
		</tr>
		<tr>
			<td>Cabin B</td>
			<td>Toilet seat</td>
			<td>33.5 (2/2)</td>
			<td>GII.1b</td>
			<td>986</td>
		</tr>
		<tr>
			<td>Lido&#160;</td>
			<td>Ice Cream Machine</td>
			<td>34.2 (2/2)</td>
			<td>GII</td>
			<td>16</td>
		</tr>
		<tr>
			<td>Lido&#160;</td>
			<td>Table Condiments</td>
			<td>35.2 (1/2)</td>
			<td>GII</td>
			<td>15</td>
		</tr>
		<tr>
			<td>Lido&#160;</td>
			<td>Table Top</td>
			<td>35.3 (1/2)</td>
			<td>GII</td>
			<td>14</td>
		</tr>
		<tr>
			<td>Pizzeria</td>
			<td>Counter surface</td>
			<td>35.7 (1/2)</td>
			<td>GII</td>
			<td>14</td>
		</tr>
		<tr>
			<td>Main Galley</td>
			<td>Fun-time Machine</td>
			<td>37.1(1/2)</td>
			<td>GII</td>
			<td>64</td>
		</tr>
		<tr>
			<td>Vending Machine</td>
			<td>Touchable Surfaces</td>
			<td>38.8 (1/2)</td>
			<td>GII</td>
			<td>18</td>
		</tr>
		<tr>
			<td>Crew lounge&#160;</td>
			<td>Keyboard Surface and Mouse</td>
			<td>36.8 (1/2)</td>
			<td>GII</td>
			<td>80</td>
		</tr>
		<tr>
			<td>Cabin C</td>
			<td>Faucet and door handle</td>
			<td>31.6 (2/2)</td>
			<td>GII.1b</td>
			<td>26,458</td>
		</tr>
		<tr>
			<td>Cabin C</td>
			<td>Telephone</td>
			<td>36.4 (2/2)</td>
			<td>GII</td>
			<td>1,035</td>
		</tr>
		<tr>
			<td>Cabin C</td>
			<td>Keyboard</td>
			<td>33.0 (2/2)</td>
			<td>GII</td>
			<td>1,317</td>
		</tr>
		<tr>
			<td>Medical center</td>
			<td>Clipboard&#160;</td>
			<td>36.0 (2/2)</td>
			<td>GII</td>
			<td>113</td>
		</tr>
		<tr>
			<td>&#160;aCabin A, B and C has been occupied by individuals who had been clinically ill with viral gastoenteristis symptoms.</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">&#160;bFour of the 17 GII-positive swab samples could be genotyped.&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">cRNA copies were caluated based on a standard curve of norovirus GII.7 RNA transcripts.&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Note: above date were slightly modified from the original article6.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Results from 34 swab samples that were collected from a cruise ship that had reported cases of suspected norovirus gastroenteritis during a voyage.
Figure 2 shows the relationship of Ct values determined by RT-qPCR and the ability to sequence these samples. In total, 127 out of 217 swab samples tested positive for GII norovirus by RT-qPCR. The samples displayed a wide range (12-40) of Ct values. In total, 29 (22.8%) of the RT-qPCR positive samples could be genotyped. Swab samples with Ct values below 27 and ranging between 28-31 were genotyped at rates of 100% and 72.2%, respectively. In contrast, only 22.0% and 1.6% of swab samples with Ct values of 32-36 and 37-40, respectively, produced hemi-nested amplicons that could be sequenced successfully.
<img alt="Figure 2" src="/files/ftp_upload/55205/55205fig2.jpg" /> 
Figure 2: Results of RT-qPCR screening and sequencing of swab samples that had been collected during confirmed norovirus outbreaks. White bars represent the number of RT-qPCR positive swab samples virus. Human norovirus nucleic acids in those RT-qPCR positive samples were amplified using hemi-nested PCR assay and then sequenced for confirmation of genotyping. Black bars and line represent the number and the percentage of genotype confirmed swab samples, respectively. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55205/55205fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ct value range</td>
			<td>Number of RT-qPCR positive samples</td>
			<td>Number (%) of sequence confirmed samples a</td>
		</tr>
		<tr>
			<td>20-23</td>
			<td>4</td>
			<td>3 (75)</td>
		</tr>
		<tr>
			<td>24-27</td>
			<td>3</td>
			<td>3 (100)</td>
		</tr>
		<tr>
			<td>28-31</td>
			<td>18</td>
			<td>13 (72.2)</td>
		</tr>
		<tr>
			<td>32-36</td>
			<td>41</td>
			<td>9 (22.0)</td>
		</tr>
		<tr>
			<td>37-40</td>
			<td>61</td>
			<td>1 (1.6)</td>
		</tr>
		<tr>
			<td colspan="3">a) hemi nested PCR&#160;</td>
		</tr>
	</tbody>
</table>
Table 2: RT-qPCR and genotyping results of swab samples.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample Number</td>
			<td>LOCATION&#160;</td>
			<td>Description of surface</td>
			<td>Surface area (cm2)</td>
			<td>Date/time of sample collection</td>
			<td>Clean (Y/N)</td>
			<td>If cleaned, what disinfectant was used?</td>
			<td>Detailed description of surface material and location&#160;</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Room# 7-1302</td>
			<td>Toilet Seat&#160;</td>
			<td>700 cm2</td>
			<td>6/26/2016; 9:00 am</td>
			<td>No</td>
			<td>not applicable</td>
			<td>Toilet seat [top surfaces), and hard plastoc</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Room #7-1330</td>
			<td>Telephone handle</td>
			<td>500 cm2</td>
			<td>6/26/2016; 9:25 am</td>
			<td>Yes</td>
			<td>1,000 ppm bleach</td>
			<td>Hard plastic, rubber button</td>
		</tr>
	</tbody>
</table>
Supplementary Table 1: Example of sample description form.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>genogroup / virus</td>
			<td>Name oligonucleotide primer/probe</td>
			<td>Sequence 5&#900;&#8594;3&#900;</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td rowspan="5">GI</td>
			<td>Cog1F</td>
			<td>CGY TGG ATG CGI TTY CAT GA</td>
			<td>17</td>
		</tr>
		<tr>
			<td>Cog1R</td>
			<td>CTT AGA CGC CAT CAT CAT TYA C</td>
			<td>17</td>
		</tr>
		<tr>
			<td>G1SKF</td>
			<td>CTG CCC GAA TTY GTA AAT GA</td>
			<td>17</td>
		</tr>
		<tr>
			<td>G1SKR</td>
			<td>CCA ACC CAR CCA TTR TAC A</td>
			<td>17</td>
		</tr>
		<tr>
			<td>Ring1E-probe</td>
			<td>FAM &#8211; TGG ACA GGR GAY CGC &#8211; MGBNFQa</td>
			<td>This study</td>
		</tr>
		<tr>
			<td rowspan="5">GII&#160;</td>
			<td>Cog2F</td>
			<td>CAR GAR BCN ATG TTY AGR TGG ATG AG</td>
			<td>17</td>
		</tr>
		<tr>
			<td>Cog2R</td>
			<td>TCG ACG CCA TCT TCA TTC ACA</td>
			<td>17</td>
		</tr>
		<tr>
			<td>Ring2-primer&#160;</td>
			<td>TGG GAG GGC GAT CGC AAT CT</td>
			<td>17</td>
		</tr>
		<tr>
			<td>G2SKR</td>
			<td>CCR CCN GCA TRH CCR TTR TAC AT</td>
			<td>17</td>
		</tr>
		<tr>
			<td>Ring2-probe</td>
			<td>Cy5 or QUASAR 670 &#8211; TGG GAG GGC GAT CGC AAT CT &#8211; BHQ2b</td>
			<td>17</td>
		</tr>
		<tr>
			<td rowspan="3">MS2</td>
			<td>MS2F</td>
			<td>TGG CAC TAC CCC TCT CCG TAT TCA CG</td>
			<td>18</td>
		</tr>
		<tr>
			<td>MS2R</td>
			<td>GTA CGG GCG ACC CCA CGA TGA C</td>
			<td>18</td>
		</tr>
		<tr>
			<td>MS2P-probe</td>
			<td>HEX - CAC ATC GAT AGA TCA AGG TGC CTA CAAGC &#8211; BHQ1c</td>
			<td>18</td>
		</tr>
		<tr>
			<td colspan="4">aGI TaqMan probe is 5&#8217;-labeled with 6-carboxyfluorescein (FAM) and 3&#8217;-labeled with MGBNFQ (Minor-groove Binding site)</td>
		</tr>
		<tr>
			<td colspan="4">bGII TaqMan probe is 5&#8217;-labeled with Cy5 or Quasar 670 and 3&#8217;-labeled with Black Hole quencher; Black Hole Quencher (BHQ) 2 used due to availability, BHQ 3 is preferred.</td>
		</tr>
		<tr>
			<td colspan="3">cMS2 TaqMan probe is 5&#8217;-labeled with HEX and 5&#8217;-labeled with BHQ1</td>
			<td></td>
		</tr>
	</tbody>
</table>
Supplementary Table 2: Oligonucleotide primers and probes information.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Volume per reaction (&#181;l)</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>2x RT-PCR Buffer*</td>
			<td>12.5</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Nuclease-free water*</td>
			<td>1.08</td>
			<td>n/a</td>
		</tr>
		<tr>
			<td>Detection Enhancer*</td>
			<td>1.67</td>
			<td>n/a</td>
		</tr>
		<tr>
			<td>Cog 1F (10 &#181;M)</td>
			<td>1</td>
			<td>400 nM</td>
		</tr>
		<tr>
			<td>Cog 1R (10 &#181;M)</td>
			<td>1</td>
			<td>400 nM</td>
		</tr>
		<tr>
			<td>Ring 1E-probe (10 &#181;M)</td>
			<td>0.5</td>
			<td>200 nM</td>
		</tr>
		<tr>
			<td>Cog 2F (10 &#181;M)</td>
			<td>1</td>
			<td>400 nM</td>
		</tr>
		<tr>
			<td>Cog 2R (10 &#181;M)</td>
			<td>1</td>
			<td>400 nM</td>
		</tr>
		<tr>
			<td>Ring 2-probe (10 &#181;M)</td>
			<td>0.5</td>
			<td>200 nM</td>
		</tr>
		<tr>
			<td>MS2F (10 &#181;M)</td>
			<td>0.25</td>
			<td>100 nM</td>
		</tr>
		<tr>
			<td>MS2R (10 &#181;M)</td>
			<td>0.25</td>
			<td>100 nM</td>
		</tr>
		<tr>
			<td>MS2P-probe (10 &#181;M)</td>
			<td>0.25</td>
			<td>100 nM</td>
		</tr>
		<tr>
			<td>2x RT-PCR enzyme *</td>
			<td>1</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Master Mix volume</td>
			<td>22</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">* included in the real time RT-PCR kit</td>
		</tr>
	</tbody>
</table>
Supplementary Table 3: Master mix for multiplex real-time GI/GII/MS2 norovirus RT-PCR
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Type of control</td>
			<td colspan="2">Result interpretation</td>
		</tr>
		<tr>
			<td rowspan="6">Sample</td>
			<td>Negative control</td>
			<td colspan="2">Should be negative</td>
		</tr>
		<tr>
			<td>Positive control</td>
			<td colspan="2">Should be positive</td>
		</tr>
		<tr>
			<td>Negative sample</td>
			<td colspan="2">Sample is negative if each Ct value is undetectable for GI/GII&#160;</td>
		</tr>
		<tr>
			<td>Positive sample</td>
			<td colspan="2">Ct values (GI/GII) of both replicates is &#60; 38; this (arbitrarily) cut-off needs to be determined experimentally for each realtime PCR platform and kit</td>
		</tr>
		<tr>
			<td>Tentative positive sample</td>
			<td>Sample is tentatively positive if Ct value (GI/GII) of one replicate is &#60;38</td>
			<td></td>
		</tr>
		<tr>
			<td>Internal process control (MS2)</td>
			<td colspan="2">Samples with a threshold cycle (Ct) value of &#8805;32 for MS2 should be retested undiluted and 1/10 diluted.</td>
		</tr>
		<tr>
			<td></td>
			<td>Parameter</td>
			<td>Acceptable value</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard curve using norovirus RNA transcripts</td>
			<td>R2</td>
			<td>&#62;0.97</td>
			<td></td>
		</tr>
		<tr>
			<td>Efficiency</td>
			<td>90% to 115%</td>
			<td></td>
		</tr>
	</tbody>
</table>
Supplementary Table 4: Controls to include in each RT-qPCR test for detection of norovirus in environmental swab samples. 
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Final concentration</td>
			<td>vol/rxn (&#181;l)</td>
		</tr>
		<tr>
			<td>5x RT-PCR buffer</td>
			<td>1x</td>
			<td>5.00</td>
		</tr>
		<tr>
			<td>dNTP mix (10 mM)</td>
			<td>0.4 mM</td>
			<td>1.00</td>
		</tr>
		<tr>
			<td>Enzyme mix (RT and Taq)</td>
			<td></td>
			<td>1.00</td>
		</tr>
		<tr>
			<td>Forward primera</td>
			<td>0.5 &#181;M</td>
			<td>0.50</td>
		</tr>
		<tr>
			<td>Reverse primer a</td>
			<td>0.5 &#181;M</td>
			<td>0.50</td>
		</tr>
		<tr>
			<td>Rnase inhibitor (20 U/&#181;l)</td>
			<td>20 U</td>
			<td>1.00</td>
		</tr>
		<tr>
			<td>Rnase-free water</td>
			<td></td>
			<td>11.00</td>
		</tr>
		<tr>
			<td>Total Volume</td>
			<td></td>
			<td>20.00</td>
		</tr>
		<tr>
			<td colspan="3">aforward and reverse primer sets for GI and GII group are Cog1F + G1SKR, and Cog2F + G2SKR, respectively.</td>
		</tr>
		<tr>
			<td>Second round RT-PCR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Final concentration</td>
			<td>vol/rxn (&#181;l)</td>
		</tr>
		<tr>
			<td>5x RT-PCR buffer</td>
			<td>1x</td>
			<td>5.00</td>
		</tr>
		<tr>
			<td>dNTP mix (10 mM)</td>
			<td>0.4 mM</td>
			<td>1.00</td>
		</tr>
		<tr>
			<td>Enzyme mix (RT and Taq)</td>
			<td></td>
			<td>1.00</td>
		</tr>
		<tr>
			<td>Forward primerb</td>
			<td>0.5 &#181;M</td>
			<td>0.50</td>
		</tr>
		<tr>
			<td>Reverse primerb</td>
			<td>0.5 &#181;M</td>
			<td>0.50</td>
		</tr>
		<tr>
			<td>Rnase inhibitor (20 U/&#181;l)</td>
			<td>20 U</td>
			<td>1.00</td>
		</tr>
		<tr>
			<td>Rnase-free water</td>
			<td></td>
			<td>14.00</td>
		</tr>
		<tr>
			<td>Total Volume</td>
			<td></td>
			<td>23.0</td>
		</tr>
		<tr>
			<td colspan="3">bforward and reverse primer sets for GI and GII group are G1SKF + G1SKR, and Ring2 primer + G2SKR, respectively.</td>
		</tr>
		<tr>
			<td>Note: above informaion was slightly modified from the original article19</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Supplementary Table 5: Hemi-nested RT-PCR Master mix.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="4">Phase I</td>
			<td rowspan="4">Field Sampling</td>
			<td>1. Check macrofoam swab kits (e.g., expiration date, tube leakages, and swab wetness)</td>
		</tr>
		<tr>
			<td>2. Swab surface (limit surface area to &#8804;100 inch2 (645 cm2))</td>
		</tr>
		<tr>
			<td>3. Place swabs into the transport tubes, and tighten the caps securely to prevent leaking during shipping</td>
		</tr>
		<tr>
			<td>4. Place swab kit in a ziplock-bag</td>
		</tr>
		<tr>
			<td rowspan="2">Phase II</td>
			<td rowspan="2">Sample transport and storage</td>
			<td>1. Sampled swabs should be stored -70 &#176;C (or -20 &#176;C)</td>
		</tr>
		<tr>
			<td>2. Transporting swab samples to laboratory at 0-4 &#176;C (cold-packs) in an insulated container and ship within 48 hr of collecting swabs</td>
		</tr>
		<tr>
			<td rowspan="4">Phase III</td>
			<td rowspan="2">RNA etraction</td>
			<td>1. Check lysis buffer (e.g., expiration data)</td>
		</tr>
		<tr>
			<td>2. Add MS2 as an internal control into lysis buffer before adding 100% ethanol</td>
		</tr>
		<tr>
			<td rowspan="2">RNA purification and concentration</td>
			<td>3. Clean lab bench and small equipment using RNA RNase removal solution</td>
		</tr>
		<tr>
			<td>4. Change gloves frequently during steps to avoid a RNA cross-contamination</td>
		</tr>
		<tr>
			<td rowspan="2">Phase IV</td>
			<td rowspan="2">RT-PCR Assay (QA/QC)</td>
			<td>1. Include quantified norovirus transcript RNAs (GI and GII) for each RT-qPCR assay to control for variations in Ct values for each PCR run</td>
		</tr>
		<tr>
			<td>2. Use absence of MS2 signal to monitor presence of PCR inhibitors</td>
		</tr>
	</tbody>
</table>
Supplementary Table 6: Checklist for CDC&#39;s environmental sampling procedure.

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 ...

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Nanyang Technological University

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

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

Environment

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 ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency&#8217;s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.

Here we present a procedure to measure total culturable viruses using the Buffalo Green Monkey kidney cell line. The procedure provides a standardized tool for measuring the occurrence of infectious viruses in environmental and drinking waters.

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. ...

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 ...

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 ...

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. 
 Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method4 which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children&#x2019;s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

Medicine

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is ...

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and&#160;seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for&#160;future pandemics.

A citizen science project was designed to recruit San Diego residents to collect environmental samples for SARS-CoV-2. A multilingual web-based platform was created for data submission using a user-friendly mobile device interface. A laboratory information management system facilitated the collection of thousands of geographically diverse samples with real-time outcome tracking.

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

Biology

An Assay to Quantify the Binding of Human Norovirus VLPs to Intestinal Bacteria

After preparing the reagents, antibodies, and bacteria as described in the manuscript, assemble all the materials in a BSL II biosafety cabinet.
Virus-like particles for human norovirus are listed as BSL II pathogens, and all work performed using VLPs should be conducted in a certified biosafety cabinet.
Add 10 micrograms of human norovirus VLPs to each tube of bacteria, and mix thoroughly by pipetting. Incubate the tubes for 1 hour at 37 degrees Celsius with constant rotation. After incubation, centrifuge the tubes at 10,000 times g for 5 minutes.
Aspirate and discard the supernatant and resuspend the bacterial pellet in 1 milliliter of PBS. After repeating the wash steps once, centrifuge the tubes again at 10,000 times g for 5 minutes. Discard the supernatant and resuspend the VLP bacteria pellet in 150 microliters of 5% blocking buffer.
A common error that occurs is that tubes are swapped and the wrong treatment is added. To prevent this, arrange all tubes in lines that correspond to the correct treatment, and add one treatment all at once to the tubes.
For each bacterial sample, prepare 50 microliters of diluted human norovirus G2 antibody. Using 5% blocking buffer, dilute the antibody 1 to 125 for E. coli case samples. Prepare 50 microliters of diluted isotype antibody for each bacterial sample using the same dilution ratios. Divide each attachment assay sample into 350-microliter aliquots by transferring them into clean 1.5-milliliter centrifuge tubes.
To create unstained controls, add 50 microliters of blocking buffer to the first aliquot from each bacterial sample. For the stained samples, add 50 microliters of the G2 antibody dilution to the second set of aliquots. Create the isotype controls by adding 50 microliters of the diluted isotype antibody to the third set of aliquots.
Incubate all the samples on ice and in the dark for 30 minutes. Then, centrifuge all the samples at 10,000 times g for 5 minutes. Discard the supernatants and resuspend each sample in 150 microliters of FCSB. Again, centrifuge all the samples at 10,000 times g for 5 minutes. Again, discard the supernatants and resuspend the samples in 100 microliters of FCSB.
After centrifuging the samples one final time and discarding the supernatant, resuspend the samples in 150 microliters of FCSB. Transfer each sample to a tube containing 400 microliters of FCSB for a total volume of 550 microliters. Store the samples at 4 degrees Celsius until they are analyzed by flow cytometry.

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

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