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).

ETICA DICHIARAZIONE: L&#39;utilizzo di avanzi di campioni clinici è approvato e segue le linee guida del Memorial Sloan Kettering Cancer Center Institutional Review Board. 1. Prima di eseguire il test NOTA: L&#39;influenza A e B test è approvato per campioni di tamponi nasofaringei e tamponi nasofaringei memorizzati nei mezzi di trasporto virale. I tamponi sono incluse nel kit e devono essere utilizzati per ottenere prestazioni ottimali. Tuttavia, rayon, schiuma, accorrevano tamponi o tamponi nasali poliestere può essere utilizzato anche per raccogliere campioni di tamponi nasali. <ol><li> Per raccogliere un campione di tampone nasale, inserire il tampone nella narice che ha il drenaggio più visibile o che è più congestionato. Con la rotazione dolce, spingere il tampone nella narice fino a incontrare resistenza e ruotare più volte contro la parete nasale prima di rimuovere lentamente dalla narice. Conservare i tamponi e trasportarli in fiale contenenti 3 ml di mezzi di trasporto virale (VTM). NOTA: Una grande attenzione al approprraccolta del campione iate deve essere preso come la raccolta dei campioni inadeguati può produrre risultati errati. NOTA: I campioni su tampone devono essere testati il ​​più presto possibile dopo la raccolta. Tuttavia, essi possono essere conservati a temperatura ambiente per un massimo di 2 ore o in frigorifero a 2-8 ° C per un massimo di 24 ore, se il test non è immediatamente disponibile. esemplari appena raccolti sono l&#39;ideale per ottenere risultati ottimali. Un uso improprio del campione, la conservazione e / o il trasporto possono produrre risultati errati. NOTA: Come misura precauzionale, gli utenti che eseguono questo test dovrebbero trattare tutti i campioni come potenzialmente infettivi seguendo le precauzioni universali durante la manipolazione dei campioni. </li><li> Portare tutti i campioni a temperatura ambiente prima del test. NOTA: Tutte le componenti di prova sono per un solo uso e non devono essere utilizzati per eseguire prove multiple. Non mescolare i componenti del kit con diverso numero di lotto. Non utilizzare i reagenti del kit oltre la data di scadenza. </li><li> Portare il ricevitore del campione azzurro di Rootemperatura m prima del test. La base di test arancione può essere provato senza la necessità di riscaldare a temperatura ambiente. </li><li> Lascia tutti i pezzi di prova nella confezione stagnola fino al momento dell&#39;uso. Accendere lo strumento premendo il pulsante di accensione sul lato dello strumento. Inserire l&#39;user-ID e premere &#39;OK&#39;. </li></ol> 2. Esecuzione del test <ol><li> Per iniziare il processo di test, toccare &#39;Esegui test&#39; sullo schermo dello strumento. Inserire l&#39;ID del paziente utilizzando la tastiera su schermo o uno scanner di codici a barre e premere &#39;OK&#39;. Aprire il coperchio e inserire la base di prova arancione nel supporto di base di test Orange. </li><li> Verificare che il criterio corretto viene visualizzato sullo schermo e premere &#39;OK&#39;. Inserire il ricevitore del campione azzurro nel supporto del ricevitore del campione azzurro. NOTA: Non aprire il ricevitore del campione prima di inserire nello strumento in modo da evitare il tampone di eluizione di raggiungere la corretta temperatura di funzionamento e può influire sulle prestazioni del test. </li&gt; <li> Attendere che il ricevitore del campione si riscaldi. Quando richiesto dallo strumento, togliere la pellicola di sigillo e inserire il tampone del paziente da testare nel ricevitore campione. NOTA: Quando si rimuove la pellicola, posizionare due dita sul bordo del ricevitore di esempio per assicurare che rimanga al suo posto. </li><li> Mescolare energicamente il tampone nel liquido per 10 secondi, premendo il capo tampone contro la parete del ricevitore campione come lo si miscela per aiutare a muovere il campione dal tampone. Premere &#39;OK&#39; per procedere. Se i tamponi di test memorizzati nel mezzo di trasporto virale, VORTEX la VTM per 10 sec quindi aggiungere 200 ml al ricevitore campione. </li><li> Premere la cartuccia di trasferimento bianco nel ricevitore del campione azzurro. Continuare a premere sul ricevitore campione fino la spia arancione aumenta. </li><li> Sollevare e quindi collegare la cartuccia di trasferimento alla base di prova. Osservare l&#39;indicatore arancione scendere una volta che il trasferimento della cartuccia è applicato correttamente. Chiudere il coperchio e non aprire fino a quando il &quot;Controllo completo&quot;Viene visualizzato un messaggio sullo schermo. </li></ol> 3. Controllo Qualità <ol><li> Toccare &#39;Run QC Test&#39; sulla schermata principale. Selezionare il test di controllo di qualità per essere eseguito. Confermare il tipo di test in modo che corrisponda il campione di controllo di qualità destinato al testing toccando &#39;OK&#39; e seguendo le istruzioni visualizzate per completare il test. </li></ol> Interpretazione 4. Risultato <ol><li> Dopo l&#39;esecuzione della prova è completa, osservare i risultati del test visualizzato sullo schermo con interpretazioni per la presenza di influenza A, B, sottotipi sconosciuti. Per ottenere risultati validi ripetere il test. </li></ol> 5. Manutenzione e pulizia <ol><li> Pulire lo strumento e la zona circostante panca ogni giorno con il 70% di etanolo o una soluzione di candeggina al 10%. Spruzzare la soluzione detergente su un panno umido, privo di lanugine per pulire. Non versare le soluzioni direttamente sullo strumento. </li></ol>

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The authors have nothing to disclose.

I virus influenzali sono importanti cause mondiali di morbilità e mortalità. diagnosi rapida e accurata di influenza è una delle principali chiavi per la gestione di focolai di influenza durante la stagione delle vie respiratorie. Altri test antigene-based sono rapidi e facili da eseguire; Tuttavia, essi hanno bassi sensibilità 13. D&#39;altra parte, i test molecolari tradizionali hanno migliorato la sensibilità, ma richiedono tecnici di laboratorio più esperti di eseguire e sono più costo...

Infezioni da virus dell&#39;influenza determinano una quantità significativa di morbilità e mortalità ogni anno 1, 2, 3. L&#39;influenza non complicata è caratterizzata da sintomi costituzionali e respiratori quali febbre, mialgia, cefalea e tosse non produttiva 4, 5. Gli individui più anziani, bambini, pazienti immunocompromessi e pazienti con comorbilità sottostanti sono a più alto rischio di complicanze gravi come la polmonite, la miocardite, malattia del sistema nervoso centrale, o la morte 6, 7. L&#39;infezione influenzale è unico da altri virus respiratori in quel tempestiva somministrazione di terapia antivirale entro 48 ore dall&#39;inizio dei sintomi può ridurre la gravità della malattia e la lunghezza 8. La rapida identificazione di influenza è stato anchedimostrato di ridurre l&#39;uso di antibiotici inutili 9, 10. Inoltre, i pazienti ospedalizzati con infezioni influenzali devono essere collocati in locali isolati con opportune precauzioni per il controllo delle infezioni. Tuttavia, le malattie respiratorie causate da virus non influenzali possono essere difficili da distinguere da clinicamente l&#39;influenza. Per questo motivo, i test diagnostici rapidi e precisi per l&#39;influenza è molto importante per la gestione clinica del paziente. Diversi test sono disponibili per il rilevamento e l&#39;identificazione di virus influenzali. Test rapidi di rilevamento dell&#39;influenza antigene (RIDTs) sono ampiamente utilizzati nella pratica clinica come test point-of-care perché sono semplici da usare e fornire i risultati entro 15 a 30 minuti 11, 12; tuttavia, la loro sensibilità variano ampiamente (10-80%) a seconda del produttore e la popolazione in fase di test, e il tipo di influenza e subtype 13, 14, 15. Saggi di fluorescenza diretta (DFA) forniscono sensibilità superiori rispetto RIDTs, ma il tempo di elaborazione è maggiore (~ 3 ore) e devono essere completati da tecnologi esperti 16, 17. Coltura virale è stato il gold standard per la diagnosi di influenza e ha migliorato la sensibilità su entrambi RIDTs e DFA 18. Tuttavia, l&#39;influenza coltura virale può richiedere 2-14 d per completare, diminuendo la sua utilità nel favorire la gestione del paziente 19. Infine, nucleico test di amplificazione degli acidi (NAAT) hanno sostituito tecniche di coltura come il nuovo gold standard nella diagnostica di influenza. NAAT sono considerati avere la massima sensibilità per rilevare l&#39;influenza in poche ore. Tuttavia, incidenza sensibile sono i test più costosi e richiedono attrezzature specializzate e tecnici di eseguire 5 <sup&gt;, 20, 21, 22, 23, 24, 25. Il test dell&#39;influenza A e B qui descritto è il primo test di influenza CLIA-rinunciato molecolare rapida che è prontamente disponibile. Questo test funziona impiegando una reazione intaccare endonucleasi Amplification (NEAR) che utilizza l&#39;amplificazione isotermica con primer specifici per l&#39;influenza seguita da rilevamento di target con sonde segnale molecolare. Questo test differenzia dell&#39;influenza A da B, richiede 2 min per impostare ed elaborare un campione, e richiede un totale di 15 minuti per completare. Qui, vi presentiamo il protocollo per il saggio Influenza A &amp; B. Inoltre, mettiamo a disposizione un insieme di dati di esempio impostare a confronto le prestazioni del virus dell&#39;influenza A e B saggio sulla archiviato tampone nasofaringeo (NPS) campioni conservati in terreno di trasporto virale (VTM) con un kit pannello patogeno respiratorio.

<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

L&#39;influenza è una malattia respiratoria contagiosa causata da virus influenzali A e B negli esseri umani e provoca una notevole quantità di morbilità e mortalità ogni anno. L&#39;influenza A e B saggio è stato il primo test molecolare rapida influenza CLIA-rinunciato a disposizione. Il test di influenza A e B funziona utilizzando l&#39;amplificazione isotermica con primer specifici per l&#39;influenza seguita da rilevamento di target con sonde segnale molecolare. Qui, le prestazioni della influenza A e B saggio su congelato, archiviati tampone nasofaringeo (NPS) campioni conservati in terreno di trasporto virale (VTM) sono stati confrontati con un test del pannello respiratoria. Le prestazioni della A e B saggio Influenza stata valutata confrontando i risultati al metodo di riferimento del pannello respiratoria. La sensibilità per il virus dell&#39;influenza A totale è stata del 67,5% (95% CI (CI), 56,6-78,5) e la specificità del 86,9% (CI, 71,0-100). Per il test virus dell&#39;influenza B, la sensibilità e la specificità sono state 90,2% (CI, 68,5-100) E il 98,8% (CI, 68,5-100), rispettivamente. Questo sistema ha il vantaggio di un tempo di prova significativamente più breve rispetto a qualsiasi altro test molecolare attualmente disponibili e la procedura semplice, priva di pipetta viene eseguito su un chiuso, sistema completamente integrato dimensioni ridotte. Nel complesso, l&#39;influenza A e dosaggio B valutati in questo studio ha il potenziale per servire come test diagnostico point-of-care rapida influenza.

In questo studio, i campioni NPS archiviati sono stati raccolti da pazienti ricoverati con sintomi simil-influenzali al Memorial Sloan-Kettering Cancer Center (MSKCC) nel corso di un focolaio di influenza tra il 15 Dicembre, 2012 e il 1 marzo 2013. I campioni NPS sono state presentate in 3 ml di VTM e testato come parte della pratica clinica di routine con un saggio molecolare che rileva un gruppo di virus respiratori (RP), tra cui l&#39;influenza a, a-1, a-3, e B. Durante il periodo di ...

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

Descriviamo un test rapido, molecolare a base di Influenza A e B. Il saggio Influenza rileva ogni bersaglio entro 15 minuti utilizzando l&#39;amplificazione isotermica con primer specifici per l&#39;influenza seguita da rilevamento di target con sonde segnale molecolare. Il saggio e influenza A B è facile da usare e richiede minimi hands-on tempo per eseguire.

Rilevazione rapida molecolare e la differenziazione dei virus influenzali A e 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 Views.  Memorial Sloan Kettering Cancer Center.  Descriviamo un test rapido, molecolare a base di Influenza A e B. Il saggio Influenza rileva ogni bersaglio entro 15 minuti utilizzando l'amplificazione isotermica con primer specifici per l'influenza seguita da rilevamento di target con sonde segnale molecolare. Il saggio e influenza A B è facile da usare e richiede minimi hands-on tempo per eseguire. 

Video: 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.

Generazione di influenza da virus ricombinante del DNA plasmidi

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

Ricerca

Immunologia e infezioni

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 rilevazione colorimetrica su un DNA microarray per l&#39;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.

Analisi quantitativa di tutti influenza di tipo A Hemagglutinins e neuraminidasi virale utilizzando anticorpi Universale di saggi semplice Slot Blot

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.

La diagnosi rapida del virus dell&#39;influenza aviaria nei volatili selvatici: l&#39;uso di un Portable rRT-PCR e liofilizzato reagenti nel campo

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 Metodo di rilevazione per il virus dell&#39;influenza

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.

Chip Fabrication Microfluidic e metodo per rilevare l&#39;influenza

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

Bioingegneria

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.

Basato sulla fluorescenza neuraminidasi Inibizione Assay per valutare la suscettibilità dei virus influenzali di neuraminidasi Inhibitor classe di antivirali

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

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.

Studi del Virus di riossidazione A in un modello murino di infezione

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.

Uso di un microarray antigeno influenzale per misurare l'ampiezza degli anticorpi del siero attraverso i sottotipi di virus

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

Saggio RT-qPCR multiplex per il rilevamento di virus respiratori

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.

Autore in primo piano: Progressi nel rilevamento multiplex dei virus respiratori 

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.

Sviluppo di saggi RT-qPCR real-time multiplex per la rilevazione di SARS-CoV-2, influenza A/B e 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 ...

Rilevazione rapida molecolare e la differenziazione dei virus influenzali A e B

Riepilogo

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Capitoli in questo video

Generazione di influenza da virus ricombinante del DNA plasmidi

ampliPHOX rilevazione colorimetrica su un DNA microarray per l'influenza

Analisi quantitativa di tutti influenza di tipo A Hemagglutinins e neuraminidasi virale utilizzando anticorpi Universale di saggi semplice Slot Blot

La diagnosi rapida del virus dell'influenza aviaria nei volatili selvatici: l'uso di un Portable rRT-PCR e liofilizzato reagenti nel campo

High-throughput Metodo di rilevazione per il virus dell'influenza

Chip Fabrication Microfluidic e metodo per rilevare l'influenza

Basato sulla fluorescenza neuraminidasi Inibizione Assay per valutare la suscettibilità dei virus influenzali di neuraminidasi Inhibitor classe di antivirali

Studi del Virus di riossidazione A in un modello murino di infezione

Uso di un microarray antigeno influenzale per misurare l'ampiezza degli anticorpi del siero attraverso i sottotipi di virus

Sviluppo di saggi RT-qPCR real-time multiplex per la rilevazione di SARS-CoV-2, influenza A/B e MERS-CoV