Name: swab sampling method for the detection of human norovirus on surfaces
Uploaded: 2017-02-06T10:45:00
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. Virol. 2 (1), 1-7 (2011).</li><li>Malek, M., 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=Outbreak+of+norovirus+infection+among+river+rafters+associated+with+packaged+delicatessen+meat,+Grand+Canyon,+2005.">Outbreak of norovirus infection among river rafters associated with packaged delicatessen meat, Grand Canyon, 2005.</a> Clin Infect Dis. 48 (1), 31-37 (2009).</li><li>Atmar, R. 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=Norwalk+virus+shedding+after+experimental+human+infection.">Norwalk virus shedding after experimental human infection.</a> Emerg. Infect. Dis. 14 (10), 1553-1557 (2008).</li><li>Glass, R. I., Parashar, U. D., Estes, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norovirus+gastroenteritis.">Norovirus gastroenteritis.</a> N. Engl. J. Med. 361 (18), 1776-1785 (2009).</li><li>Park, G. W., 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+New+Environmental+Sampling+Protocol+for+Detection+of+Human+Norovirus+on+Inanimate+Surfaces.">Evaluation of a New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate Surfaces.</a> Appl. Environ. Microbiol. 81 (17), 5987-5992 (2015).</li><li>Barker, J., Jones, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+spread+of+infection+caused+by+aerosol+contamination+of+surfaces+after+flushing+a+domestic+toilet.">The potential spread of infection caused by aerosol contamination of surfaces after flushing a domestic toilet.</a> J. Appl. Microbiol. 99, 339-347 (2005).</li><li>Tung-Thompson, G., Libera, D. A., Koch, K. L., de Los Reyes, F. L., Jaykus, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerosolization+of+a+Human+Norovirus+Surrogate,+Bacteriophage+MS2,+during+Simulated+Vomiting.">Aerosolization of a Human Norovirus Surrogate, Bacteriophage MS2, during Simulated Vomiting.</a> PloS one. 10, 0134277 (2015).</li><li>Atmar, R. 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=Determination+of+the+50%+human+infectious+dose+for+Norwalk+virus.">Determination of the 50% human infectious dose for Norwalk virus.</a> J. Infect. Dis. 209 (7), 1016-1022 (2014).</li><li>Petrignani, M., van Beek, J., Borsboom, G., Richardus, J. H., Koopmans, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norovirus+introduction+routes+into+nursing+homes+and+risk+factors+for+spread:+a+systematic+review+and+meta-analysis+of+observational+studies.">Norovirus introduction routes into nursing homes and risk factors for spread: a systematic review and meta-analysis of observational studies.</a> J. Hosp. Infect. 89 (3), 163-178 (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centers+for+Disease+Control+Prevention.+Norovirus+outbreak+in+an+elementary+school--District+of+Columbia,+February+2007.">Centers for Disease Control Prevention. Norovirus outbreak in an elementary school--District of Columbia, February 2007.</a> MMWR. Morb. Mortal. Wkly. Rep. 56 (51-52), 1340-1343 (2008).</li><li>Cheesbrough, J. S., Barkess-Jones, L., Brown, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+prolonged+environmental+survival+of+small+round+structured+viruses.">Possible prolonged environmental survival of small round structured viruses.</a> J. Hosp. Infect. 35, 325-326 (1997).</li><li>Julian, T. R., Tamayo, F. J., Leckie, J. O., Boehm, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+surface+sampling+methods+for+virus+recovery+from+fomites.">Comparison of surface sampling methods for virus recovery from fomites.</a> Appl. Environ. Microbiol. 77, 6918-6925 (2011).</li><li>Taku, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+and+detection+of+caliciviruses+from+food+contact+surfaces.">Concentration and detection of caliciviruses from food contact surfaces.</a> J. Food. Prot. 65, 999-1004 (2002).</li><li>Scherer, K., Ellerbroek, L., Schulenburg, J., Johne, R., Klein, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+swab+sampling+method+for+the+detection+of+norovirus+and+rotavirus+on+artifically+contaminated+food+and+environmental+surfaces.">Application of a swab sampling method for the detection of norovirus and rotavirus on artifically contaminated food and environmental surfaces.</a> Food. Environ. Virol. 1 (42), 42-49 (2009).</li><li>Herzog, A. B., 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+sample+recovery+efficiency+for+bacteriophage+P22+on+fomites.">Evaluation of sample recovery efficiency for bacteriophage P22 on fomites.</a> Appl. Environ. Microbiol. 78, 7915-7922 (2012).</li><li>Vega, 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=CaliciNet:+A+Novel+Surveillance+Network+for+Norovirus+Gastroenteritis+Outbreaks+in+the+United+States.">CaliciNet: A Novel Surveillance Network for Norovirus Gastroenteritis Outbreaks in the United States.</a> Emerging Infectious Diseases. 17 (8), 1389-1395 (2011).</li><li>Rolfe, K. 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. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+Surface+Sampling+and+Recovery+of+Viruses+and+Non-Spore+Forming+Bacteria+on+a+QMRA+Model+for+Fomites.">The Effect of Surface Sampling and Recovery of Viruses and Non-Spore Forming Bacteria on a QMRA Model for Fomites.</a> Environ. Sci. Technol. 50 (11), 5945-5952 (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiology+of+food+and+animal+feed-Horizontal+method+for+determination+of+hepatitis+A+virus+and+norovirus+in+food+using+real-time+RT-PCR.">Microbiology of food and animal feed-Horizontal method for determination of hepatitis A virus and norovirus in food using real-time RT-PCR.</a> International Organization for Standardization (ISO). , (2013).</li><li>Huslage, K., Rutala, W. A., Sickbert-Bennett, E., Weber, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+approach+to+defining+"high-touch"+surfaces+in+hospitals.">A quantitative approach to defining "high-touch" surfaces in hospitals.</a> Infect. Control. Hosp. Epidemiol. 31 (8), 850-853 (2010).</li><li>Wu, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+norovirus+outbreak+at+a+long-term-care+facility:+the+role+of+environmental+surface+contamination.">A norovirus outbreak at a long-term-care facility: the role of environmental surface contamination.</a> Infect. Control. Hosp. Epidemiol. 26 (10), 802-810 (2005).</li><li>Ikner, L. A., Gerba, C. P., Bright, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+and+recovery+of+viruses+from+water:+a+comprehensive+review.">Concentration and recovery of viruses from water: a comprehensive review.</a> Food Environ. Virol. 4 (2), 41-67 (2012).</li><li>Gallimore, C. I., 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=Environmental+monitoring+for+gastroenteric+viruses+in+a+pediatric+primary+immunodeficiency+unit.">Environmental monitoring for gastroenteric viruses in a pediatric primary immunodeficiency unit.</a> J. Clin. Microbiol. 44 (2), 395-399 (2006).</li><li>Ganime, A. C., 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=Dissemination+of+human+adenoviruses+and+rotavirus+species+A+on+fomites+of+hospital+pediatric+units.">Dissemination of human adenoviruses and rotavirus species A on fomites of hospital pediatric units.</a> Am J Infect Control. , (2016).</li><li>Verani, M., Bigazzi, R., Carducci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+contamination+of+aerosol+and+surfaces+through+toilet+use+in+health+care+and+other+settings.">Viral contamination of aerosol and surfaces through toilet use in health care and other settings.</a> Am J Infect Control. 42 (7), 758-762 (2014).</li></ol>

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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		<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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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 ...

The overall goal of our macrofoam based surface sampling method is to identify outbreak etiology, overall evaluation of environmental contamination dynamics of the virus, and effectiveness of remediation strategies for the norovirus. This protocol detailed how to collect swab sample from environmental surface and how these swabs should be stored and transported, and which laboratory protocol are recommended for the detection and typing of the norovirus from these swabs. Clear improvements of this environmental swab protocol for noroviruses is that we have determined the best type of swab material, how much norovirus can be recovered from hard surfaces, and that the protocol can be used effectively to sample larger surface areas.In this video, two of my staff scientists, Geun Woo Park and and Preeti Chhabra will demonstrate the swab protocol for the detection and typing of noroviruses. To begin swabbing, proceed to the sampling area and move the swab across the sampling area with one stroke in the horizontal direction, one stroke in the vertical direction, and one stroke in the diagonal direction. Place each swab into a separate tube and tighten the cap securely.Sample surfaces with stainless steel, sample a toilet seat surface, and sample a doorknob. Next, label one 15-milliliter tube and one RNA Midi column for each sample. Include a negative extraction control in each experiment.Prepare an empty bottle to collect universal nucleic acid extraction or UNEX, buffer waste. To prepare the lysis solution for 10 swabs, mix 25 milliliters of UNEX buffer with 25 milliliters of PBST. Add 50 microliters of coliphage MS2 suspension to 50 milliliters of lysis solution.Place a swab in a 15-milliliter tube, and add five milliliters of lysis solution. Mix the swab in the solution, and incubate the mixture at room temperature. Add five milliliters of 100%ethanol to each tube, and vortex the tubes for 10 seconds.Carefully remove the swab from the 15-milliliter tube, pressing it gently against the side of the tube to remove excess liquid, and then discard the swab. Carefully transfer 4.5 milliliters of the UNEX ethanol mixture onto a Midi column, and centrifuge the column. After centrifugation, discard the filtrate.Load another 4.5 milliliters from the same mixture onto the same column, then centrifuge the column. Discard the filtrate. For the first wash, add 3.5 milliliters of 70%ethanol onto the Midi column, and centrifuge the column.After the spin, discard the filtrate. For the second wash, add another 3.5 milliliters of 70%ethanol onto the Midi column. Centrifuge and discard the filtrate.For the dry spin, centrifuge the Midi columns to remove all traces of ethanol. Place the Midi column in a new 15-milliliter centrifuge tube. Add 250 microliters of elution buffer onto the Midi column, and wait for one minute before centrifuging to obtain the highest to viral RNA recovery.Add 500 microliters of RNA binding buffer to 250 microliters of RNA, and vortex the mixture for 10 seconds. Add 750 microliters of 100%ethanol, and vortex for 10 seconds. Next, label a spin column for each sample.Load the 750-microliter sample onto a spin column, and centrifuge the spin columns. Discard the flow-through. Load the remaining 750 microliters onto a spin column, and centrifuge the columns.For the prewash, add 400 microliters of RNA prep buffer to each spin column, and centrifuge the column. Discard the flow-through. For the first wash, add 800 microliters of RNA wash buffer to each spin column, and centrifuge the columns.Discard the flow-through. For the second wash, add 400 microliters of RNA wash buffer to each spin column, and centrifuge them. Discard the flow-through.For the dry spin, centrifuge the spin column to remove all traces of wash buffer. Carefully transfer the spin column to a clean 1.5-milliliter microcentrifuge tube. Add 25 microliters of elution buffer onto the spin column, and incubate the column for one minute at room temperature to increase viral RNA recovery from the column.Collect the RNA by centrifuging the spin column at 10, 000 times G for one minute. Proceed directly to real-time PCR. Before continuing with multiplex real-time RTPCR, clean working surfaces, pipettes, and centrifuges with an RNase decontamination solution.Thaw real-time PCR reagents on ice. Thaw the sample RNA on ice in a separate area. Determine the number of reactions, and prepare at least 10%more reagent for the master mix to control for losses during pipetting.Next, vortex the individual master mix components for five seconds. Carefully mix the enzyme by flicking the tube. Briefly centrifuge the master mix components for five seconds.Then, prepare the master mix for the RTPCR detection of norovirus G1 and G2 according to the kit instructions, and add norovirus-specific oligonucleotide primers and probes in a 1.5-milliliter microcentrifuge tube. Next, mix the master mix by pipetting seven times up and down. Aliquot 22 microliters of the master mix into each well of the real-time PCR plate.Vortex the sample RNA for five seconds, and collect the contents by five-second centrifugation. Add three microliters of sample RNA positive controls and 10-fold serial dilutions of G1 and G2 RNA transcripts to the real-time PCR plate. Add three microliters of nuclease-free water into the node template control, or NTC wells.Seal the plate with optical adhesive film. Carefully centrifuge the plate at 1, 300 times G for one minute to remove any air bubbles or liquid drops that may be present in the wells. Then, set up the real-time PCR, and enter the thermocycling conditions.Determine the standard curves for both the G1 and the G2 transcripts. Convert the Ct values into RNA copies using the respective standard curve. Take the ratio of the volume of total RNA eluent and the total amount of RNA used for the real-time assay to calculate the total number of RNA copies per sample.To genotype the norovirus-positive samples using hemi-nested conventional RTPCR, first prepare a master mix for the first round RTPCR, and dispense 20-microliter aliquots in pre-labeld PCR tubes. Add five microliters of RNA to each tube. After the first round RTPCR is completed, prepare a master mix for the hemi-nested second PCR round by dispensing aliquots of 23 microliters into new tubes.Finally, add two microliters of the first round PCR product to each tube. The macrofoam-based swab sampling method was field tested on samples collected from a cruise ship that had reported cases of suspected norovirus gastroenteritis. Four of the 17 norovirus-positive swab samples could be genotyped, and all samples had identical G2 sequences.Viral load determination allowed assessment of surfaces which were heavily contaminated, such as toilet seats. The relationship of cycle threshold, or Ct values, was determined by real-time RTPCR in the ability to sequence these samples. In total, 127 out of 217 swab samples tested positive for G2 norovirus.Swab samples with Ct values between 24 and 27 and between 28 and 31 were genotyped at rates of 100%and 72.2%respectively. In contrast, only 22%and 1.6%of swab samples with Ct values between 32 and 36 and 37 to 40 were sequenced successfully. As we have demonstrated, this protocol allows to determine the level of environmental contamination during norovirus outbreaks, detect the possible cause of an outbreak when clinical samples are not available, and also, to determine the effectiveness of cleaning practices.For the most accurate result, it is very important to follow this protocol precisely to minimize the variation of the results among the different samples.

Research

JoVE Journal

Immunology and Infection

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

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 ...

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 ...

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 ...

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, ...

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 ...

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 ...

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

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 ...

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 ...

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

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 ...