Name: rapid molecular detection and differentiation of influenza viruses a and b
Uploaded: 2017-01-30T14:00:00.000+00:00
Duration: 5 min 38 s
Description: Watch this Scientific Journal Video about Rapid Molecular Detection and Differentiation of Influenza Viruses A and B at JoVE.com

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>

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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><tbody><tr><td>Alere i Instrument</td><td>Alere</td><td>NAT-000 (Global), NAT-024 (US)</td><td></td></tr><tr><td>Alere i Influenza A &amp;amp; B 24 Test Kit</td><td>Alere</td><td>425-000 (Global), 425-024 (US)</td><td></td></tr><tr><td>Alere i Barcode Scanner</td><td>Alere</td><td>EQ001001</td><td></td></tr><tr><td>Alere Universal Printer</td><td>Alere</td><td>55115 (Global), alereiprinter&amp;nbsp;(US)</td><td></td></tr><tr><td>200 &amp;micro;l precision pipette</td><td></td><td></td><td></td></tr><tr><td>200 &amp;micro;l disposable pipette tips</td><td></td><td></td><td></td></tr><tr><td>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

15.6K 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

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Erzeugung von rekombinanten Influenza Virus von Plasmid-DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

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 ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Quantitative Analysen aller Influenza Typ A Viral Hämagglutininen und Neuraminidasen mit Universal Antikörper in Einfache Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

Schnelle Diagnose der Aviären Influenza Virus bei Wildvögeln: Verwendung eines tragbaren rRT-PCR und gefriergetrocknet Reagenzien in das Feld

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

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, ...

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

Mikrofluidischen Chip Herstellung und Verfahren zur Influenza Detect

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

Biotechnik

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Influenza-A-Virus-Studien in einem Mausmodell der Infektion

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens.
This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera.
Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

We present a protocol for using a protein microarray constructed by printing influenza antigens onto nitrocellulose-coated slides to simultaneously probe serum for multiple antibody isotypes against over 250 antigens from different virus strains, thus allowing measurement of the breadth of serum antibodies across virus subtypes.

Verwendung eines Influenza-Antigen-Mikroarrays zur Messung der Breite von Serum-Antikörpern über Virussubtypen hinweg

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge ...

Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System

Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of A. baumannii is crucial for prompt treatment, effective infection control, and curbing antibiotic resistance. However, there is no suitable method for rapid and easy on-site detection of A. baumannii. The DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) system offers a rapid, precise, and sensitive approach to A. baumannii detection by integrating the target-specific recognition capabilities of Cas12a with the isothermal amplification efficiency of Recombinase Polymerase Amplification (RPA). This protocol details the detection of A. baumannii using RPA combined with LbaCas12a endonuclease. The following steps are described in this article: extraction of DNA, selection of a specific DNA sequence, design of primer and CRISPR RNA (crRNA), construction of positive recombinant plasmid, setup of Cas12a-RPA assay, optimization of the RPA amplification system, visualization of the RPA-CRISPR/Cas12a assay using a fluorescence detection tool such as a real-time PCR instrument, and evaluation of sensitivity and specificity evaluation.

To facilitate the rapid and precise detection of Acinetobacter baumannii, we present a protocol that employs Recombinase Polymerase Amplification (RPA) in conjunction with LbaCas12a endonuclease for identifying A. baumannii infections.

Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of ...

Multiplex RT-qPCR-Assay zum Nachweis von Atemwegsviren

Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general public&#39;s health. During influenza seasons, the spread of SARS-CoV-2 and other respiratory viruses may cause a population-wide burden of respiratory disease that is difficult to manage. For that, the respiratory viruses SARS-CoV-2, Influenza A, Influenza B, and Middle East respiratory syndrome (MERS-CoV) will need to be carefully watched over in the upcoming fall and winter seasons, particularly in the case of SARS-CoV-2, Influenza A, and Influenza B, which share similar epidemiological factors like susceptible populations, mode of transmission, and clinical syndromes. Without target-specific assays, it can be challenging to differentiate among cases of these viruses owing to their similarities. Accordingly, a sensitive and targeted multiplex assay that can easily differentiate between these viral targets will be useful for healthcare practitioners. In this study, we developed a real-time reverse transcriptase-PCR-based assay utilizing an in-house developed R3T one-step RT-qPCR kit for simultaneous detection of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV. With as few as 10 copies of their synthetic RNAs, we can successfully identify SARS-CoV-2, Influenza A, Influenza B, and MERS-CoV targets simultaneously with 100% specificity. This assay is found to be accurate, reliable, simple, sensitive, and specific. The developed method can be used as an optimized SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

Autor im Rampenlicht: Fortschritte bei der Multiplex-Detektion von Atemwegsviren 

We present two probe-based in-house one-step RT-qPCR kits for common respiratory viruses. The first assay is for SARS-CoV-2 (N), Influenza A (H1N1 and H3N2), and Influenza B. The second is for SARS-Cov-2 (N) and MERS (UpE and ORF1a). These assays can be successfully implemented in any specialized laboratory.

Entwicklung von Multiplex-Real-Time-RT-qPCR-Assays zum Nachweis von SARS-CoV-2, Influenza A/B und MERS-CoV

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general ...