Name: swab sampling method for the detection of human norovirus on surfaces
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Description: Watch this Scientific Journal Video about Swab Sampling Method for the Detection of Human Norovirus on Surfaces at JoVE.com

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 directi...

<ol>
	<li>Isakbaeva, E. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norovirus+transmission+on+cruise+ship.">Norovirus transmission on cruise ship.</a> Emerg. Infect. Dis. 11, 154-158 (2005).</li><li>Lopman, B. A., Gasta&#241;aduy, P., Park, G. W., Hall, A. J., Parashar, U. D., Vinj&#233;, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+transmission+of+norovirus+gastroenteritis.">Environmental transmission of norovirus gastroenteritis.</a> Curr. Opin. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+internally+controlled,+one-step,+real-time+RT-PCR+assay+for+norovirus+detection+and+genogrouping.">An internally controlled, one-step, real-time RT-PCR assay for norovirus detection and genogrouping.</a> J Clin Virol. 39 (4), 318-321 (2007).</li><li>Kittigul, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norovirus+GII-4+2006b+variant+circulating+in+patients+with+acute+Thailand+during+a+2006-2007+study.">Norovirus GII-4 2006b variant circulating in patients with acute Thailand during a 2006-2007 study.</a> J. Med. Virol. 82 (5), 854-860 (2010).</li><li>Teunis, P. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norwalk+virus:+how+infectious+is+it.">Norwalk virus: how infectious is it.</a> J. Med. Virol. 80 (8), 1468-1476 (2008).</li><li>Wollants, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+norovirus+sampling+method+using+sodium+dodecyl+sulfate/EDTA-pretreated+chromatography+paper+strips.">Evaluation of a norovirus sampling method using sodium dodecyl sulfate/EDTA-pretreated chromatography paper strips.</a> J. Virol. Methods. 122, 45-48 (2004).</li><li>Weir, M. H., Shibata, T., Masago, Y., Cologgi, D., Rose, J. 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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.

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	<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>
	</tbody>
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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 ...

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