Name: Swab Sampling Method for the Detection of Human Norovirus on Surfaces (Video) | JoVE Journal
Uploaded: 2017-02-06T10:45:00.000+00:00
Duration: 10 min 3 s
Description: Learn macrofoam swab sampling for detecting human norovirus on environmental surfaces. This protocol improves virus recovery rates and enables effective outbreak investigation and contamination monitoring.

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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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</ol>

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

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

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

<table><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

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="Nucleic acid extraction process diagram; RT-PCR setup for norovirus detection and genotyping." src="/files/ftp_upload/55205/55205fig1.jpg" data-altupdatedat="17...

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.

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

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