We thank the Clinical Microbiology Service staff of the Memorial Sloan-Kettering Cancer Center for help in collecting clinical specimens. This study was supported in part by a research agreement between MSKCC and Alere Scarborough (SK2013-0262).

ETHIK STATEMENT: Die Verwendung von übrig gebliebenen klinischen Proben zugelassen ist und nach den Richtlinien des Memorial Sloan Kettering Cancer Center Institutional Review Board. 1. Vor dem Assay Lauf HINWEIS: Das Influenza-A-und B-Test wird für Nasen-Rachen-Abstrichproben und für Nasen-Rachen-Abstrichen in viralen Transportmedien gespeichert zugelassen. Tupfer sind im Kit enthalten und sollte für eine optimale Leistung verwendet werden. Jedoch, Rayon, Schaum, beflockte Tupfer oder Polyester Nasenabstrichen können auch Nasentupferproben zu sammeln, verwendet werden. <ol><li> Um eine Nasenabstrichprobe sammeln, legen Sie den Tupfer in das Nasenloch, die am meisten sichtbaren Drainage oder das ist am stärksten belasteten. Mit sanften Drehung, drücken Sie den Tupfer in das Nasenloch bis auf Widerstand stoßen und mehrmals gegen die Nasenwand drehen, bevor sie aus dem Nasenloch langsam entfernen. Lagern Sie die Tupfer und transportieren sie in Fläschchen mit 3 ml Virustransportmedien (VTM). HINWEIS: Eine sorgfältige Aufmerksamkeit auf appropriate Probenentnahme muss als unzureichend Probenentnahme entnommen werden kann, zu falschen Ergebnissen führen. HINWEIS: Abstrichproben sollten so bald wie möglich nach der Entnahme getestet werden. Sie können jedoch bei Raumtemperatur für bis zu 2 Stunden oder gekühlt bei 2-8 ° C bis zu 24 Stunden aufbewahrt werden, wenn der Test nicht sofort verfügbar ist. Frisch sind gesammelt Proben für eine optimale Testleistung ideal. Eine unsachgemäße Handhabung von Proben, Lagerung und / oder Transport können zu fehlerhaften Ergebnissen führen. HINWEIS: Als Vorsichtsmaßnahme, Einzelpersonen der Durchführung dieser Prüfung sollten alle Proben zu behandeln, als potenziell infektiös durch allgemeine Vorsichtsmaßnahmen Folgendes, wenn Umgang mit den Proben. </li><li> Bringen Sie alle Proben auf Raumtemperatur vor dem Test. HINWEIS: Alle Testkomponenten für den einmaligen Gebrauch sind nur und sollte nicht verwendet werden, um mehrere Tests durchzuführen. Kitkomponenten nicht unterschiedlicher Chargen mischen. Nicht Kitreagenzien nach dem Verfallsdatum nicht mehr verwenden. </li><li> Bringen Sie den blauen Probeaufnahme zu room Temperatur vor der Prüfung. Die orange Testbasis kann ohne die Notwendigkeit, auf Raumtemperatur erwärmen, getestet werden. </li><li> Lassen Sie alle Teststücke in der Folienverpackung erst unmittelbar vor dem Gebrauch. Schalten Sie das Gerät durch Drücken der Power-Taste an der Seite des Gerätes. Geben Sie die User-ID ein und drücken Sie &quot;OK&quot;. </li></ol> 2. Durchführung des Tests <ol><li> Um den Testprozess zu beginnen, berühren &#39;Run Test &quot;auf der Instrumentenanzeige. Geben Sie die Patienten-ID, indem Sie auf dem Bildschirm, Tastatur oder Barcode-Scanner ein und drücken Sie &quot;OK&quot;. Öffnen Sie den Deckel und legen Sie die orange Testbasis in den Testgrundhalter orange. </li><li> Bestätigen Sie, dass der richtige Test auf dem Bildschirm angezeigt wird, und drücken Sie &quot;OK&quot;. Legen Sie die blauen Probeaufnahme in den blauen Probenaufnahmehalter. HINWEIS: Schalten Sie den Proben Empfänger nicht öffnen, bevor sie in das Gerät legen, da dies die Elutionspuffer verhindern die richtige Betriebstemperatur erreicht und können die Testleistung auswirken. </li&gt; <li> Warten Sie auf die Probeaufnahme, um sich aufzuwärmen. Wenn vom Gerät aufgefordert, die Foliendichtung entfernen und den Patiententupfer setzen in die Probenaufnahmeeinrichtung getestet werden. HINWEIS: Wenn die Folie zu entfernen, zwei Finger auf den Rand des Sample-Empfänger, um sicherzustellen, dass es an seinem Platz bleibt. </li><li> Energisch den Tupfer für 10 Sekunden in der Flüssigkeit mischen, wird der Tupfer gegen die Seite durch Drücken der Probenaufnahme, wie Sie es mischen, um die Probe aus dem Tupfer entfernen. Drücken Sie auf &quot;OK&quot;, um fortzufahren. Wenn die Prüfung in viralen Transportmedium gespeichert Tupfer, fügen Sie die VTM für 10 sec vortexen dann 200 &amp; mgr; l der Probe Empfänger. </li><li> Drücken Sie die weiße Transferpatrone in den blauen Probeaufnahme. Weiter auf der Probeaufnahme drücken, bis die orangefarbene Anzeige steigt. </li><li> Heben und dann die Transfereinheit an der Testbasis verbinden. Beachten Sie die orangefarbene Anzeige absteigen, wenn die Patrone Übertragung korrekt angebracht ist. Den Deckel schließen und nicht erst öffnen, wenn der &quot;Test Complete&quot;Meldung erscheint auf dem Bildschirm. </li></ol> 3. Qualitätskontrolle <ol><li> Tippen Sie auf &quot;Run QC Test &#39;auf dem Home-Bildschirm. Wählen Sie den QC-Test zu starten. Bestätigen Sie den Testtyp der QC-Probe für den Test vorgesehen übereinstimmen durch Berühren &quot;OK&quot;, und im Anschluss an die auf dem Bildschirm Tests abgeschlossen auffordert. </li></ol> 4. Ergebnisauswertung <ol><li> Nachdem der Testlauf abgeschlossen ist, beachten Sie die Ergebnisse des Tests auf dem Bildschirm mit Interpretationen auf die Anwesenheit von Influenza A, B, unbekannte Subtypen angezeigt. Für ungültige Ergebnisse wiederholen Sie den Test. </li></ol> 5. Wartung und Reinigung <ol><li> Reinigen Sie das Gerät und die umliegende Bankbereich täglich mit 70% Ethanol oder 10% Bleichlösung. Sprühen Sie die Reinigungslösung auf einem feuchten, fusselfreien Tuch zu reinigen. Gießen Sie die Lösungen nicht direkt auf dem Instrument. </li></ol>

<ol>
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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=The+Underrecognized+Burden+of+Influenza+in+Young+Children.">The Underrecognized Burden of Influenza in Young Children.</a> New England Journal of Medicine. 355 (1), 31-40 (2006).</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=Estimates+of+Deaths+Associated+with+Seasonal+Influenza+---+United+States,+1976-2007.">Estimates of Deaths Associated with Seasonal Influenza --- United States, 1976-2007.</a> MMWR. 59 (33), 1057-1089 (2010).</li><li>Agrawal, A. S., 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=Comparative+evaluation+of+real-time+PCR+and+conventional+RT-PCR+during+a+2+year+surveillance+for+influenza+and+respiratory+syncytial+virus+among+children+with+acute+respiratory+infections+in+Kolkata,+India,+reveals+a+distinct+seasonality+of+infection.">Comparative evaluation of real-time PCR and conventional RT-PCR during a 2 year surveillance for influenza and respiratory syncytial virus among children with acute respiratory infections in Kolkata, India, reveals a distinct seasonality of infection.</a> Journal of Medical Microbiology. 58 (12), 1616-1622 (2009).</li><li>Ginocchio, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strengths+and+Weaknesses+of+FDA-Approved/Cleared+Diagnostic+Devices+for+the+Molecular+Detection+of+Respiratory+Pathogens.">Strengths and Weaknesses of FDA-Approved/Cleared Diagnostic Devices for the Molecular Detection of Respiratory Pathogens.</a> Clinical Infectious Diseases. 52, 312-325 (2011).</li><li>Grohskopf, L. 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=Prevention+and+control+of+seasonal+influenza+with+vaccines:+recommendations+of+the+Advisory+Committee+on+Immunization+Practices-United+States,+2013-2014.">Prevention and control of seasonal influenza with vaccines: recommendations of the Advisory Committee on Immunization Practices-United States, 2013-2014.</a> MMWR Recomm Rep. 62 (07), 1-43 (2013).</li><li>Molinari, N. -. A. 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=The+annual+impact+of+seasonal+influenza+in+the+US:+measuring+disease+burden+and+costs.">The annual impact of seasonal influenza in the US: measuring disease burden and costs.</a> Vaccine. 25 (27), 5086-5096 (2007).</li><li>Aoki, F. Y., 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=Early+administration+of+oral+oseltamivir+increases+the+benefits+of+influenza+treatment.">Early administration of oral oseltamivir increases the benefits of influenza treatment.</a> Journal of Antimicrobial Chemotherapy. 51 (1), 123-129 (2003).</li><li>Noyola, D. E., Demmler, G. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+six+influenza+rapid+tests+in+detecting+human+influenza+in+clinical+specimens.">Performance of six influenza rapid tests in detecting human influenza in clinical specimens.</a> Journal of Clinical Virology. 39 (2), 132-135 (2007).</li><li>Fiore, A. 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=Antiviral+agents+for+the+treatment+and+chemoprophylaxis+of+influenza+---+recommendations+of+the+Advisory+Committee+on+Immunization+Practices+(ACIP).">Antiviral agents for the treatment and chemoprophylaxis of influenza --- recommendations of the Advisory Committee on Immunization Practices (ACIP).</a> MMWR Recomm Rep. 60 (1), 1-24 (2011).</li><li>Harper, S. 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=Seasonal+influenza+in+adults+and+children--diagnosis,+treatment,+chemoprophylaxis,+and+institutional+outbreak+management:+clinical+practice+guidelines+of+the+Infectious+Diseases+Society+of+America.">Seasonal influenza in adults and children--diagnosis, treatment, chemoprophylaxis, and institutional outbreak management: clinical practice guidelines of the Infectious Diseases Society of America.</a> Clin Infect Dis. 48 (8), 1003-1032 (2009).</li><li>Hannoun, C., Tumova, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+on+influenza+laboratory+diagnostic+and+surveillance+methods+in+Europe.">Survey on influenza laboratory diagnostic and surveillance methods in Europe.</a> European Journal of Epidemiology. 16 (3), 217-222 (2000).</li><li>Leonardi, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+of+2009+H1N1+influenza+A+virus+using+fluorescent+antibody+methods.">Rapid identification of 2009 H1N1 influenza A virus using fluorescent antibody methods.</a> Am J Clin Pathol. 134 (6), 910-914 (2010).</li><li>Chartrand, C., Leeflang, M. M. G., Minion, J., Brewer, T., Pai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+Rapid+Influenza+Diagnostic+TestsA+Meta-analysis.">Accuracy of Rapid Influenza Diagnostic TestsA Meta-analysis.</a> Annals of Internal Medicine. 156 (7), 500-511 (2012).</li><li>Lee, G. 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M., Elkan, M., Leet, M., McGowan, K. L., Hodinka, R. 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J., Selvarangan, R. <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+the+Alere+I+influenza+A&amp;B+nucleic+acid+amplification+test+by+use+of+respiratory+specimens+collected+in+viral+transport+medium.">Evaluation of the Alere I influenza A&amp;B nucleic acid amplification test by use of respiratory specimens collected in viral transport medium.</a> J Clin Microbiol. 52 (11), 3992-3995 (2014).</li><li>Nie, S., 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+Alere+i+Influenza+A&amp;B+for+Rapid+Detection+of+Influenza+Viruses+A+and+B.">Evaluation of Alere i Influenza A&amp;B for Rapid Detection of Influenza Viruses A and B.</a> Journal of Clinical Microbiology. 52 (9), 3339-3344 (2014).</li><li>Chapin, K. C., Flores-Cortez, E. 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The authors have nothing to disclose.

Influenzaviren sind signifikant weltweit Ursachen von Morbidität und Mortalität. Eine schnelle und genaue Diagnose der Influenza ist eine der wichtigsten Schlüssel zur Grippe-Ausbrüche während Atem Saison zu verwalten. Andere Antigen-basierte Tests sind schnell und einfach durchzuführen; sie haben jedoch geringe Empfindlichkeiten 13. Auf der anderen Seite haben die traditionellen molekularen Tests Empfindlichkeit verbessert, benötigen aber mehr erfahrenen Labortechniker durchführen und si...

Influenza - Virusinfektionen führen zu einer signifikanten Menge von Morbidität und Mortalität jedes Jahr 1, 2, 3. Unkompliziert Influenza wird durch Verfassungs- und respiratorische Symptome wie Fieber, Muskelschmerzen, Kopfschmerzen, und nicht produktiven Husten 4, 5 aus. Ältere Menschen, Kleinkinder, immungeschwächten Patienten und Patienten mit zugrunde liegenden Komorbiditäten sind zu einem höheren Risiko für schwere Komplikationen wie Lungenentzündung, Myokarditis, Zentralnervensystem - Krankheit oder Tod 6, 7. Influenza - Infektion ist einzigartig von anderen respiratorischen Viren in diesem Prompt Verabreichung von antiviralen Therapie innerhalb von 48 Stunden nach Einsetzen der Symptome kann die Schwere der Erkrankung und der Länge 8 zu reduzieren. Schnelle Identifikation von Influenza auch gewesen9, 10 gezeigt , die Verwendung von Antibiotika unnötig zu verringern. Ferner hospitalisierten Patienten mit Influenza-Infektionen müssen in isolierten Räumen mit entsprechenden Infektionsschutzmaßnahmen gesetzt werden. Jedoch durch nicht-Influenzaviren verursacht Atemwegserkrankungen kann schwierig sein, klinisch von Influenza unterscheiden. Aus diesem Grunde ist eine schnelle und genaue diagnostische Tests für Influenza sehr wichtig für die klinische Behandlung der Patienten. Mehrere Tests sind für den Nachweis und die Identifizierung von Influenza-Viren zur Verfügung. Schnelle Influenza - Antigen - Nachweistests (RIDTs) sind weit verbreitet in der klinischen Praxis als Point-of-Care - Tests verwendet , weil sie einfach zu bedienen sind und liefern Ergebnisse innerhalb von 15 bis 30 min 11, 12; jedoch variieren ihre Empfindlichkeiten stark (10-80%) in Abhängigkeit von dem Hersteller und der Population getestet, und die influenza Typ und SUBTY13 pe, 14, 15. Direkte Fluoreszenzassays (DFAs) eine überlegene Empfindlichkeit gegenüber RIDTs, aber die Verarbeitungszeit größer (~ 3 h) , und muss von erfahrenen Technikern 16, 17 abgeschlossen werden. Viral Kultur hat für Influenza - Diagnostik der Gold - Standard und hat eine verbesserte Empfindlichkeit über beide RIDTs und DFAs 18. Allerdings Influenzaviruskultur kann überall 2-14 d in Anspruch nehmen, ihre Nützlichkeit verringern in 19 Patienten - Management unterstützen. Schließlich haben die Nukleinsäureamplifikation Tests (NAAT) Kulturtechniken wie der neue Goldstandard in der Influenza-Diagnostik ersetzt. NAAT werden als zum Detektieren influenza in wenigen Stunden die größte Empfindlichkeit zu haben. NAAT sind jedoch die teuersten Assays und erfordern spezielle Ausrüstung und Technologen 5 auszuführen <snach oben&gt;, 20, 21, 22, 23, 24, 25. Das Influenza-A-und B-Test hier beschrieben wird, ist die erste CLIA-verzichtet Molekular schnelle Grippe-Test, die leicht verfügbar ist. Dieser Test funktioniert durch eine Nicking Endonuklease Amplifikationsreaktion (NEAR) unter Anwendung, die durch Zielerfassung mit molekularen Signalsonden gefolgt isothermen Amplifikation mit Influenza-spezifischen Primer verwendet. Dieser Test unterscheidet Influenza A von B, erfordert 2 Minuten einzurichten und eine Probe verarbeiten und erfordert insgesamt 15 Minuten zu beenden. Hier präsentieren wir das Protokoll für die Influenza A &amp; B-Test. Darüber hinaus bieten wir ein Beispieldaten die Leistung des Influenza-A-und B-Test auf archivierten Nasen-Rachen-Abstrich (NPS) Exemplare, gespeichert in der viralen Transportmedium zu vergleichen (VTM) zu einem anderen respiratorischen Erreger Panel-Untersuchung.

<table border="0" cellpadding="0" cellspacing="0" width="660">
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	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">Alere i Instrument</td>
			<td style="width:64px;">Alere</td>
			<td style="width:303px;">NAT-000 (Global), NAT-024 (US)</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alere i Influenza A &#38; B 24 Test Kit</td>
			<td>Alere</td>
			<td style="width:303px;">425-000 (Global), 425-024 (US)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alere i Barcode Scanner</td>
			<td>Alere</td>
			<td>EQ001001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alere Universal Printer</td>
			<td>Alere</td>
			<td style="width:303px;">55115 (Global), alereiprinter&#160;(US)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">200 &#181;L precision pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">200 &#181;L disposable pipette tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Viral transport medium</td>
			<td>Remel</td>
			<td>M4-RT</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid molecular detection and differentiation of influenza viruses a and b

Influenza ist eine ansteckende durch Influenzaviren A und B in Menschen verursacht respiratorische Erkrankung und verursacht eine erhebliche Menge an Morbidität und Mortalität jedes Jahr. Das Influenza-A-und B-Test war der erste CLIA-verzichtet Molekular schnelle Grippe-Test zur Verfügung. Die Influenza A- und B-Test funktioniert durch Verwendung isothermen Amplifikation mit Influenza-spezifischen Primern durch die Zielerfassung mit molecular beacon Sonden folgten. Hier wird die Leistung des Influenza-A-und B-Test auf gefrorenen archivierten Nasen-Rachen-Abstrich (NPS) Proben in Virustransportmedium (VTM) gespeichert wurden einem Atem Panel-Untersuchung verglichen. Die Leistung des Influenza A- und B-Assay wurde durch Vergleich der Ergebnisse der Atemtafel Referenzmethode bewertet. Die Empfindlichkeit für die gesamte Influenza-Virus A betrug 67,5% (95% CI (CI), 56,6-78,5) und eine Spezifität von 86,9% (CI, 71,0 bis 100). Für B-Tests Influenza-Virus, die Sensitivität und Spezifität waren 90,2% (CI, 68,5 bis 100) Und 98,8% (CI, 68,5 bis 100), respectively. Dieses System hat den Vorteil einer erheblich kürzeren Prüfzeit als alle anderen derzeit verfügbaren molekularen Assays und der einfachen, Pipettenfreien Verfahren läuft auf einer voll integrierten, geschlossenen, mit kleinem Platzbedarf System. Insgesamt hat das Influenza-A-und B-Test in dieser Studie untersuchte das Potenzial als Point-of-Care schnelle Influenza diagnostischen Test zu dienen.

In dieser Studie wurden archivierten NPS Proben von Patienten stationär gesammelt 15, am Memorial Sloan-Kettering Cancer Center (MSKCC) während einer Influenza-Ausbruch zwischen Dezember mit grippeähnlichen Symptomen präsentiert 2012 und 1. März 2013. Die NPS Exemplare in 3 ml vorgelegt wurden von VTM und als Teil der klinischen Routine mit einem molekularen Test getestet, die eine Gruppe von Atemwegsviren (RP), einschließlich Influenza A, A-1, A-3 und B. Während der Studie, 3675 ...

Watch this Scientific Journal Video about Rapid Molecular Detection and Differentiation of Influenza Viruses A and B at JoVE.com

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Wir beschreiben eine schnelle, molekülbasierten Influenza A und B-Assay. Die Influenza-Test weist jedes Ziel innerhalb von 15 Minuten durch den Einsatz von isothermen Amplifikation mit Influenza-spezifischen Primer durch Zielerfassung mit molekularen Signalsonden gefolgt. Das Influenza-A-und B-Test ist benutzerfreundlich und benötigt minimale Hands-on-Zeit auszuführen.

Schnelle molekulare Erkennung und Differenzierung von Influenza-Viren A und B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

rapid-molecular-detection-differentiation-influenza-viruses

Research

JoVE Journal

Immunology and Infection

15.5K Aufrufe.  Memorial Sloan Kettering Cancer Center.  Wir beschreiben eine schnelle, molekülbasierten Influenza A und B-Assay. Die Influenza-Test weist jedes Ziel innerhalb von 15 Minuten durch den Einsatz von isothermen Amplifikation mit Influenza-spezifischen Primer durch Zielerfassung mit molekularen Signalsonden gefolgt. Das Influenza-A-und B-Test ist benutzerfreundlich und benötigt minimale Hands-on-Zeit auszuführen. 

Serie: Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

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

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, ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Schnelle Analyse von Chromosomenaberrationen in Maus-B-Lymphozyten durch PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

Der Nachweis von IgE-Wahre exprimierenden Maus B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Die intranasale Verabreichung von rekombinantem Influenza Vaccines in chimären Maus-Modelle zu Mucosale Immunität Studieren

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Nasal Tücher für Influenza-A-Virus-Erkennung und Isolierung von Schweine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

Detektion und Anreicherung von Rare Antigen-spezifische B-Zellen für die Analyse von Phänotypen und Funktion

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

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 ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Messung der Influenza neutralisierenden Antikörperantworten gegen a/H3N2-Viren im menschlichen Serum von Microneutralization Assays mit MDCK-SIAT1 Zellen

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...