The authors have no acknowledgements.

1. Swab Probenahme im Feld <ol><li> Tragen Sie ein sauberes Paar Handschuhe. </li><li> Messen Sie die Größe des Beprobungsfläche ohne Berührung der Oberfläche mit einem Maßband oder Lineal. Versuchen Sie, den Bereich so genau wie möglich zu ermitteln und füllen Sie ein Berichtsformular (Supplementary Tabelle 1). </li><li> Überprüfen Sie den Tupfer-Kit für mögliche Leckagen und Etikettenprobentransporttaschen und Tupfer-Kits. </li><li> Bewegen des Tupfers über die Flächenabtastung folgendermaßen: einen Hub in horizontaler Richtung, einen Hub in vertikaler Richtung und einem Hub in einer diagonalen Richtung. Seien Sie nicht eine Fläche größer als 700 cm 2 Tupfer. </li><li> Legen Sie jedes Tupfer in ein Röhrchen und ziehen Sie die Kappe. </li></ol> 2. Lagerung und Transport von Putzlappen zum Labor <ol><li> Halten Tupfer bei 4 ° C für bis zu 48 h. Falls eine Lagerung für einen längeren Zeitraum erforderlich ist, lagern die Abstriche bei -20 ° C (oder -70 ° C). </li><li> Halten Sie die Röhrchen bei 0-4 ° C (dh. verwenden, Kältepackungen) in einem isolierten Behälter während des Transports zum Labor und werden innerhalb von 24 bis 48 h der Sammlung. </li></ol> 3. Viruskonzentration, Viral RNA Extraktion und Reinigung HINWEIS: Alle Zentrifugationsschritte verwenden, um eine Tischzentrifuge bei 5.000 × g für 5 Minuten bei Raumtemperatur, sofern nicht anders angegeben. Seien Sie besonders vorsichtig, wenn Sie mit der universellen Nukleinsäure-Extraktion (UNEX) Puffer arbeiten. Schutzbrille oder Gesichtsschutz. <ol><li> Beschriften Sie ein 15 ml-Röhrchen und einer RNA-Midi-Säule für jede Probe. Fügen Sie eine negative Extraktionskontrolle in jedem Experiment. </li><li> Die Lyse-Lösung für 10 Tupfer vorzubereiten, mit 25 ml PBST (PBS, pH 7,2, enthaltend 0,02% Tween-80) 25 ml UNEX Puffer mischen. </li><li> Je 50 ul der Coliphage MS2 - Suspension (10 &amp; sup6 ; PFU / &amp; mgr; l) zu 50 ml Lysis - Lösung. </li><li> Legen Sie einen Tupfer in einem 15 ml-Röhrchen und mit 5 ml Lyselösung. Mischen und Inkubation für 10 min bei Raumtemperatur. </li><li>5 ml 100% Ethanol zu jedem Röhrchen und Vortex für 10 s. </li><li> Vorsichtig den Tupfer aus dem 15 ml-Röhrchen zu entfernen (es enthält UNEX Puffer) es leicht gegen die Seite des Schlauchpress überschüssige Flüssigkeit zu entfernen und dann den Tupfer verwerfen. Die restlichen Mengen sollten zwischen 9-10 ml sein. </li></ol> 4. Midi Säule viralen Nukleinsäureextraktion <ol><li> Transfer vorsichtig 4,5 ml UNEX / Ethanol-Mischung aus Schritt 3.6 auf einem Midi-Säule, Zentrifuge und Filtrat verwerfen. </li><li> Legen Sie eine andere 4,5 ml aus der gleichen Mischung auf die gleiche Säule, Zentrifuge und verwerfen Sie das Filtrat. </li><li> Für die erste Wäsche, fügen Sie 3,5 ml 70% Ethanol auf die Midi-Säule, Zentrifuge und das Filtrat verwerfen. </li><li> Für die zweite Wäsche, weitere 3,5 ml 70% Ethanol auf die Midi-Spalte hinzufügen. Zentrifuge und Filtrat verwerfen. </li><li> Für die Trockenschleuder Zentrifugation der Midi Spalten alle Spuren von Ethanol zu entfernen (was sich negativ auf Ihre virale RNA-Gewinnung beeinflussen können). </li><li>Legen Sie die Midi-Spalte in einer neuen 15 ml Zentrifugenröhrchen. In 250 ul Elutionspuffer auf die Midi-Säule; 1 min warten, bevor das Zentrifugieren der höchsten viralen RNA-Gewinnung zu erhalten. </li><li> Die extrahierten Nukleinsäure bei -70 ° C oder sofort zu Schritt 5. </li></ol> 5. Die Konzentration der viralen Nukleinsäure Verwendung von RNA-Clean and Concentrator Kits <ol><li> In 500 &amp; mgr; l RNA Binding Buffer zu 250 &amp; mgr; l in Schritt eluierte RNA 4.7 oben und Vortex für 10 s. </li><li> In 750 &amp; mgr; l 100% Ethanol und Vortex für 10 s. </li><li> Beschriften Sie eine Spin-Säule für jede Probe. Laden Sie die 750 &amp; mgr; l Probe auf eine Spin-Säule. </li><li> Zentrifugieren Sie die Spin-Säulen bei 12.000 × g für 1 min. Verwerfen Sie den Durchfluss durch. </li><li> Legen Sie die restlichen 750 &amp; mgr; l auf eine Spin-Säule und Zentrifuge bei 12.000 g für 1 min. </li><li> Für die Vorwäsche, werden 400 &amp; mgr; l RNA Prep Puffer zu jeder Spin-Säule und Zentrifuge bei 12.000 g für 1 min. Verwerfen Sie den Durchfluss durch. </li><li> Für die erste Wäsche, fügen 800 &amp; mgr; l RNA Waschpuffer in jede Spin-Säule und Zentrifuge bei 12.000 g für 1 min. Verwerfen Sie den Durchfluss durch. </li><li> Für das zweite Waschen werden 400 &amp; mgr; l RNA Waschpuffer Säule und Zentrifuge bei 12.000 g für 1 min zu spinnen. Verwerfen Sie den Durchfluss durch. </li><li> Für die Trockenschleuder Zentrifugation der Spin-Säule alle Spuren von Waschpuffer für 2 min bei 12.000 xg zu entfernen. </li><li> Transfer vorsichtig Spin-Säule in ein sauberes 1,5 ml Mikrozentrifugenröhrchen. </li><li> Zugeben von 25 &amp; mgr; l Elutionspuffer auf die spin-Säule und Inkubation für 1 min bei Raumtemperatur. Dadurch wird die Rückgewinnung von viraler RNA aus der Säule zu erhöhen. </li><li> Sammeln RNA durch die Spin-Säule bei 10.000 × g für 1 min zentrifugiert. </li><li> Entweder gehen Sie direkt auf RT-qPCR (Schritt 6) oder Speicher-RNA bei -70 ° C. </li></ol> 6. Mutiplex RT-qPCR Nachweis von Genogruppe I und II Noroviren und Coliphage MS 2 (Supplementary Tabelle 2) <ol><li> Saubere Arbeitsflächen, pipettes und Zentrifugen mit RNase Dekontaminationslösung auf eine mögliche Kontamination zu reduzieren. </li><li> Auftauen RT-qPCR-Reagenzien auf Eis. Thaw RNA auf Eis (in einem separaten Bereich / Zimmer). </li><li> Bestimmen Sie die Anzahl der Reaktionen und fügen Sie mindestens 10% mehr von ihnen (zB wenn 10 Reaktionen benötigt werden, machen Master - Mix für 11). </li><li> Vortex einzelnen Master-Mix-Komponenten, mit Ausnahme von 25x RT-qPCR Enzym, für 5 s. durch Ausklopfen der Röhre mit einem Finger vorsichtig das Enzym mischen. </li><li> Kurz Zentrifuge Master-Mix-Komponenten für 5 s. </li><li> Bereiten Sie den Master - Mix für die Echtzeit - RT-PCR - Nachweis von Noroviren GI und GII gemäß dem Kit - Anweisungen und Norovirus-spezifischen Oligonukleotid - Primer und Sonden in einem 1,5 ml Mikrozentrifugenröhrchen (Supplementary Tabelle 3) hinzuzufügen. </li><li> Mischen Sie den Master-Mix durch Pipettieren von 5-10 mal nach oben und unten (Verwirbelung wird nicht empfohlen). Aliquot 22 ul des Master-Mix in jeder Vertiefung der Echtzeit-PCR-96-Well-Platte. </li><li> Vortex Probe RNA5 Sekunden, und sammeln Sie durch kurzes (5 s) Zentrifugation. </li><li> In 3 ul der Probe RNA und GI und GII positive Kontrollen an den RT-qPCR Platte (folgen Vorlage aus Tabelle 2). In 3 ul Nuklease-Freewater in den No-Template-Steuerung (NTC) Brunnen. </li><li> Verschließen Sie die Echtzeit-Platte mit optischen Klebefilm. </li><li> Zentrifuge vorsichtig die Echtzeit-Platte bei 1300 × g für 1 min alle Luftblasen oder Flüssigkeitstropfen zu entfernen, die in den Vertiefungen vorhanden sein können. </li><li> Stellen Sie eine Echtzeit-PCR-Instrument und Set-up folgende Thermozyklisierung Bedingungen: 1) RT-Schritt 10 min. bei 45 ° C (2) Aktivierung der Taq-Polymerase, 10 min. bei 95 ° C und (3) 45 Zyklen von 15 s bei 95 ° C und 60 s bei 60 ° C. </li></ol> 7. Quantifizierung von Norovirus in Tupferproben <ol><li> Überprüfen Sie die Ergebnisse der positiven und negativen Kontrollen RT-qPCR Ergebnisse (Ergänzende Tabelle 3) zu validieren. </li><li> Bestimmen Sie, ob jede Standardkurve erfüllt dieakzeptable Werte in Ergänzungs Tabelle 2 und berechnen die Gesamtstandardabweichung für die Standardkurve unter Verwendung von Gleichung 1 gegeben. (Gleichung 1)% Wirkungsgrad = 100 x 10 (Durchschnitt CT - Werte-Intercept) / Slope. </li><li> Wenn die PCR - Thermocycler - Software wird die Steigung nicht für jede Standardkurve zu berechnen, bestimmen Steigung und R 2 Werte , die durch Regression Statistik - Software (zB Excel, SPSS oder SAS). Darüber hinaus berechnen für Standardkurven folgende Gleichung I die% Wirkungsgrad </li><li> Notieren Sie sich die RNA - Kopienzahl von Thermocycler - Software berechnet für alle Testproben auf der Basis von Standardkurven, die den Kriterien in Ergänzende Tabelle 4 erfüllen. Rerun alle Proben mit Standard - t Hut Kurven erfüllen nicht die Kriterien oder haben falsch-positive Kontrollen. Überprüfen Ct-Werte von Coliphage MS2, die als interne Kontrolle zur Überwachung PCR Hemmung aufgenommen. </li><li> Bestimmen Sie die Gesamtzahl der RNA-Kopien per Probe durch die in Schritt 7.2 berechnet, um die RNA-Kopienzahl multipliziert mit dem Verhältnis des Volumens (25 bis 50 &amp; mgr; l) der gesamten RNA Eluent derjenigen der RNA (3 bis 5 &amp; mgr; l), die für die RT-qPCR-Reaktion. Alternativ berechnen die RNA - Dichte durch die Gesamt - RNA - Kopienzahl durch die Objektfläche (cm 2) analysiert geteilt wird . </li></ol> 8. Genotypisierung von Echtzeit-RT-PCR-positiven Proben von Hemi Nested Herkömmliche PCR-Amplifikation <ol><li> Bereiten Sie die erste Runde des Master - Mix für jeden Primersatz der RT-PCR - Assays (Supplementary Tabellen 2 und 5). </li><li> In 5 ul Norovirus positive RNA zu 20 &amp; mgr; l Master-Mix. </li><li> Führen Sie die RT-PCR unter den folgenden Bedingungen: (1) RT-Schritt 30 min bei 42 ° C (2) Aktivierung der Taq-Polymerase, 15 min bei 95 ° C, und (3) 40 Zyklen von 30 s bei 95 ° C , 30 s bei 50 ° C und 60 sec bei 72 ° C. Nach 40 Zyklen bei 72 ° C für weitere 10 min inkubiert. </li><li>Bereiten Sie die zweite Runde der mater - Mix für jeden Primersatz der RT-PCR - Assays (Supplementary Tabellen 2 und 5). </li><li> In 23 &amp; mgr; l Master-Mix und mit 2 &amp; mgr; l der ersten Runde RT-PCR-Produkte (von der ersten Runde) zu jedem Röhrchen (idealerweise der ersten Runde Produkt 1/10 in RNase-freiem Wasser verdünnt werden sollte). </li><li> Wiederholen Sie Schritt 8.3. </li><li> Vorbereiten eines 2% igen Agarose-Gel in 100 ml 1x Tris-Acetat-EDTA (40 mM Tris, 20 mM Essigsäure und 1 mM EDTA bei pH 8,3). Nach dem Auflösen von Agarose, indem die Mischung in einem Mikrowellenofen erhitzt wird, 10 ul Nukleinsäurefarbstoff pro 100 ml der vorbereiteten Gel hinzu, nachdem die Mischung auf 60-70 ° C abgekühlt, um das Gel gießen und die Kämme einzufügen. Lassen Sie das Gel für mindestens 30 min absetzen. </li><li> Mischen Sie 15 ul jeder RT-PCR-Produkt mit 3 &amp; mgr; l 6-fachem Beladungs ​​Farbstoff. Last 15 ul jeder Probe und dem Agarosegel elektrophoretisch bei 100 V für 1 h. </li><li> Excise Ziel RT-PCR-Fragmente von geeigneter Größe (330 bp für GI und 341 bp für GII) aus dem Gel eind RNA unter Verwendung eines kommerziellen Gel-Extraktions-Kits reinigen. Das gereinigte PCR-Produkt kann jetzt Sanger-Sequenzierung verwendet werden. </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.

Noroviren haben eine 50% human infektiöse Dosis zwischen 18 und 10 drei Viruspartikel 20. Deshalb, auch Low-Level-Kontamination von Oberflächen kann eine Gefahr für die Gesundheit darstellen. Verschiedene Aspekte der Tupfer Probenahmeprotokoll wurden einschließlich ausgewertet: 1) verschiedene Tupfer Materialien, 2) Lagerbedingungen Tupfer während des Transports, 3) Virus-RNA-Konzentration und 4) Coliphage MS2 als interne Extraktionskontrolle. Bis vor ...

<html> Menschliche Noroviren sind eine führende Ursache für Epidemie und sporadischen akuten Gastroenteritis weltweit 1, 2, 3. Das Virus ist sehr ansteckend und Übertragung erfolgt durch direkten Mensch zu Mensch Interaktion oder indirekt durch Kontakt mit kontaminierten Lebensmitteln, Wasser oder Umwelt-Oberflächen. Noroviren können für längere Zeit vergossen werden und verlängert das Überleben des Virus auf Umwelt Oberflächen hat 1 dokumentiert, 2, 3. Während der Spitzenabwurf, Milliarden von Viruspartikeln pro Gramm Faeces freigesetzt werden, und Erbrochenem enthält auch eine ausreichende Anzahl von Viruspartikeln 4 Infektion zu verursachen, 5, 6, 7, 8,ef &quot;&gt; 9, 10. Darüber hinaus wird die Übertragung des Virus zwischen leblosen Oberflächen und die menschliche Haut kann leicht 2, 11, 12. Daher Überwachung der Umweltverschmutzung in Ausbruchsuntersuchungen auftreten unterstützen können und in der Wirksamkeit der Clean-up - Bewertung und Desinfektionsverfahren. Mehrere Umweltprobenahme Protokolle zum Nachweis von Rotavirus, Coliphage MS2, feline Calicivirus (FCV) und Bakteriophagen P22 13, 14, 15, 16 beschrieben worden. Jedoch sind die Validierungsbedingungen in diesen Studien beschrieben, einschließlich der schnellen Austrocknung (&lt;1 h) und kleine Oberflächenbereiche (25 x 100 cm 2), möglicherweise nicht ausreichend Feldeinstellungen repräsentieren. Darüber hinaus ist die geringe Belastung von Enviro erwartetnmental Oberflächen erfordern Protokolle, die der Lage sind, sehr wenige Viruspartikel zu detektieren. Wir entwickelten eine Makroschaum-basierte Oberfläche Probenahmeverfahren zum Nachweis und zur Typisierung von Norovirus. Dieses Verfahren wurde bei mehreren Norovirusausbrüchen validiert. Das Protokoll enthält 1), wie Tupferproben von Umwelt Oberflächen (2), wie die Integrität der Proben während der Entnahme und Versand an das Labor, und 3) Labortests und Typisierung von Norovirus am besten halten zu sammeln.

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			<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>
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		<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

Menschliche Noroviren sind eine führende Ursache für Epidemie und sporadische Gastroenteritis weltweit. Da die meisten Infektionen entweder direkt über die Person-to-Person Route oder indirekt durch Umwelt Oberflächen oder Lebensmittel übertragen werden, kontaminierte fomites und leblose Oberflächen sind wichtige Vehikel für die Verbreitung des Virus während Norovirusausbrüchen. Wir entwickelt und evaluiert ein Protokoll Makroschaums Tupfer zum Nachweis und zur Typisierung von humanen Noroviren von harten Oberflächen verwendet wird. Im Vergleich zu faser bestückte Tupfer oder antistatische Wischtücher ermöglichen Makroschaum Tupfer Virus Recovery (Bereich 1,2 bis 33,6%) von Toilettensitzflächen von bis zu 700 cm 2. Das Protokoll enthält die Schritte zur Extraktion des Virus aus den Tupfer und eine weitere Konzentration der viralen RNA unter Verwendung von Spin-Säulen. Insgesamt 127 (58,5%) von 217 Tupferproben, die von den Oberflächen auf Kreuzfahrtschiffen und Langzeitpflegeeinrichtungen, wo Norovirus-Gastroenteritis war gesammelt worden warenpositiv getestet GII Norovirus durch RT-qPCR berichtet. Von diesen 29 (22,8%) erfolgreich genotypisiert werden konnte. Abschließend Nachweis von Noroviren auf Umgebungsoberflächen unter Verwendung des Protokolls wir bei der Bestimmung des Grades der Umweltverschmutzung während der Ausbrüche sowie Nachweis von Virus kann helfen entwickelt, wenn klinische Proben nicht zur Verfügung stehen; es kann auch die Überwachung der Wirksamkeit von Sanierungsstrategien erleichtern.

Abbildung 1 zeigt ein Flussdiagramm des Tupferprobenahmeprotokoll. Dieses Protokoll besteht aus vier Hauptschritten; 1) Probenentnahme, 2) Probenlagerung und Transport, 3) die virale RNA-Reinigung und Konzentration und 4) RT-qPCR-Assay und die Genotypisierung. <img alt="Abbildung 1" src="/files/ftp_upload/55205/55205fig1.jpg" /> Abbildung 1: Ablaufdiagramm des endgültigen Protokolls für Umweltob...

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.

Swab Sampling Verfahren zum Nachweis von humanem Norovirus auf Oberflächen

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

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

Research

JoVE Journal

Immunology and Infection

19.5K Aufrufe.  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. 

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

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

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

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

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Kombination von Klebstoff-tape-basierten Sampling und Fluoreszenz In situ Hybridisierung zum schnellen Nachweis von Salmonellen 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 ...

Forschung

Immunologie und Infektion

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.

Finger-Stick Blutprobenentnahme Methodik zur Bestimmung der Übung-induzierte Lymphozyten Apoptose

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

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

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

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

ampliPHOX kolorimetrischen Nachweis einer DNA Microarray für 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, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

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

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

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

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

Super-Resolution Imaging der natürlichen Killerzell Immunologische Synapse auf einem Glas-gestützten Planar Lipiddoppelschicht

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

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

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

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

Qualitative und quantitative Assays zur Detektion und Charakterisierung von Protein Antimikrobielle

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

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

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

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

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

Magnetrührers Verfahren zum Nachweis von<em&gt; Trichinella</em&gt; Larvae in Muskelproben

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

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

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

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

Fluoreszenz-basierte Neuraminidase-Inhibitionsassay die Empfänglichkeit der Influenza-Viren an die Neuraminidase-Inhibitor-Klasse von Anti-Virus zu beurteilen

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

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

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

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

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

Nicht-invasive Probenahme von Schleimhaut-Flüssigkeit für die Quantifizierung von In Vivo oberen Atemwegen immun-Mediator-Level

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