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).

ETİK TABLOSU: kullanımı sol üzerinde klinik örneklerin onaylanmış ve Memorial Sloan Kettering Kanser Merkezi Kurumsal Değerlendirme Kurulu kuralları izler. 1. Testi Koşu önce NOT: İnfluenza A ve B tahlil nazofarengeal sürüntü örneklerinin ve viral taşıma medya saklanan nazofarengeal sürüntü için onaylanmıştır. Svaplar kit dahil edilir ve en iyi performans için kullanılmalıdır. Ancak, suni ipek, köpük, çubuklar veya polyester burun bezlerden de nazal sürüntü örnekleri toplamak için kullanılabilir akın etti. <ol><li> bir nazal sürüntü örneği toplamak için, en görünür drenaj vardır ya da en sıkışık burun deliğine çubukla yerleştirin. Direnç sağlanana kadar nazik rotasyon ile, burun deliğine çubukla itin ve yavaşça burun deliğinden çıkarmadan önce burun duvara birkaç kez çevirin. bezlerden saklayın ve viral taşıma medya (VTM) 3 ml&#39;lik şişelerde taşırlar. NOT: Dikkatli dikkat appropr içiniate örnek toplama hatalı sonuçlar verebilir yetersiz örnek toplama olarak alınmalıdır. NOT: Swab örnekleri toplandıktan sonra en kısa sürede test edilmelidir. Bununla birlikte, en çok 2 saat oda sıcaklığında muhafaza veya test şimdi kullanılabilir değilse kadar 24 saat boyunca 2-8 ° C&#39;de soğutulmaktadır. Taze toplanan örnekler, testin performansı için idealdir. Yanlış numune işleme, depolama ve / veya taşıma hatalı sonuçlar doğurabilir. NOT: Bir önlem olarak, bu testi bireylerin örneklerini tutarken evrensel önlemler takip ederek potansiyel olarak bulaşıcı tüm örnekleri tedavi gerekir. </li><li> Test öncesinde, oda sıcaklığına kadar tüm örnekler getirin. Not: Tüm test bileşenleri, sadece tek bir kullanım için ve birden fazla testlerini yapmak için kullanılmamalıdır. Farklı lot numaralarına kit bileşenlerini karıştırmayın. son kullanma tarihi geçmiş kit reaktifleri kullanmayın. </li><li> roo mavi örnek alıcı getirTest öncesinde m sıcaklığı. Turuncu test tabanı, oda sıcaklığına kadar ısınmaya gerek kalmadan test edilebilir. </li><li> kadar kullanılmadan hemen önce folyo sarma tüm test parçaları bırakın. Cihazın yan tarafında bulunan güç düğmesine basarak alet açın. Kullanıcı kimliği ve Enter tuşuna basın &#39;Tamam.&#39; </li></ol> 2. Testi Koşu <ol><li> Test sürecine başlamak için, enstrüman ekranda &#39;Run Testi&#39; dokunun. Ekran klavyesini veya barkod tarayıcı ve basın kullanılarak hastaların kimliğini girin &#39;OK.&#39; Kapağı açın ve Portakal testi baz tutucu içine Portakal testi tabanını yerleştirin. </li><li> doğru test ekranı ve basın görüntülendiğini onaylayın &#39;OK.&#39; Mavi numune alıcı tutucu içine mavi örnek alıcısını takın. NOT: Bu doğru çalışma sıcaklığını ulaşmasını elüsyon tamponu engeller ve test performansını etkileyebilir olarak enstrüman yerleştirmeden önce örnek alıcısı açmayın. </li&gt; <li> Örnek alıcı ısınmak için bekleyin. enstrüman tarafından istendiğinde, folyo mühür kaldırmak ve örnek alıcı içine test edilecek hasta bezi yerleştirin. NOT: folyo sökerken, yerinde kalmasını sağlamak için örnek Alıcı kenarında iki parmağınızı yerleştirin. </li><li> Şiddetle size çubukla gelen numune çıkarmak yardımcı karıştırarak olarak numune alıcı tarafında karşı çubukla kafasına basarak, 10 saniye boyunca sıvı içinde çubukla karıştırın. Basın &#39;Tamam&#39; devam etmek. viral taşıma ortamında saklanan test bezlerden, 10 saniye boyunca VTM vorteks sonra numune alıcısına 200 ul ekleyin. </li><li> mavi numune alıcı içine beyaz aktarım kartuşunu basın. turuncu gösterge yükselir kadar örnek alıcısı üzerinde basmaya devam edin. </li><li> Kaldırın ve daha sonra test tabanına aktarma kartuşu takın. Kartuş transferi doğru bağlandığından kez turuncu gösterge gözlemleyin inerler. Kapak kapatın ve &quot;Test Complete&quot; kadar açmayınmesajı ekranda görünür. </li></ol> 3. Kalite Kontrol <ol><li> Ana ekranda &#39;Run QC Testi&#39; dokunun. QC Testi seçmek Run edilecek. &#39;Tamam&#39; dokunarak ve ekrandaki testi tamamlamak ister takip ederek test amaçlı QC örnek eşleşecek test türünü onaylamak. </li></ol> 4. Sonuç Yorumlama <ol><li> test çalıştırması tamamlandıktan sonra, İnfluenza A, B, bilinmeyen alt tiplerin varlığı için yorumlarıyla ekranda test sonuçlarını gözlemleyin. Geçersiz sonuçlar için testi tekrarlayın. </li></ol> 5. Bakım ve Temizlik <ol><li> alet ve% 70 etanol ya da% 10 çamaşır suyu solüsyonu ile günlük olarak çevredeki tezgah temizleyin. temizlemek için nemli, tüy bırakmayan bir bez üzerine temizleme solüsyonu püskürtün. enstrüman üzerinde doğrudan çözüm dökmeyin. </li></ol>

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</ol>

The authors have nothing to disclose.

Grip virüsleri morbidite ve mortalite dünya çapında nedenlerdir. grip hızlı ve doğru tanı, solunum sezonunda grip salgınları yönetmek için en önemli anahtarlarından biridir. Diğer antijen temelli testler, hızlı ve gerçekleştirmek kolaydır; ancak, düşük duyarlılıkları 13 var. Öte yandan, geleneksel moleküler testler hassasiyeti daha iyi, ancak yapmak ve daha pahalı olan daha deneyimli laboratuar teknologlar ihtiyaç var. Bu çalışma ve protokol açıklanan Grip A ve ...

Grip virüsü enfeksiyonları morbidite ve mortalite, her yıl 1, 2, 3, önemli bir miktarda sonuçlanır. Komplike olmayan grip, ateş, kas ağrısı, baş ağrısı ve kuru öksürük 4, 5 olarak anayasal ve solunum semptomları ile karakterizedir. Altta yatan eşlik eden yaşlı bireyler, küçük çocuklar, immün sistemi baskılanmış hastalar ve hasta pnömoni, miyokardit, merkezi sinir sistemi hastalığı veya ölüm 6, 7 gibi ciddi komplikasyonlara için daha yüksek risk altındadır. Grip enfeksiyonu hastalığın şiddetini ve süresini 8 azaltabilir semptom başlangıcından 48 saat içinde antiviral tedavinin bu istemi yönetiminde diğer solunum yolu virüsleri benzersizdir. grip hızlı tespiti de olmuşturGereksiz antibiyotik 9, 10 kullanımını azalttığı gösterilmiştir. Ayrıca, grip enfeksiyonu olan yatan hastalar, uygun enfeksiyon kontrol önlemleri ile izole oda yerleştirilmesi gerekir. klinik grip ayırt etmek Ancak, sigara grip virüslerinin neden olduğu solunum yolu hastalıkları zor olabilir. Bu nedenle, grip için hızlı ve doğru tanı testi, klinik hasta yönetimi için çok önemlidir. Çeşitli deneyler tespiti ve grip virüslerinin tanımlanması için kullanılabilir. Kullandıkları ve 15 dakika 11, 12 ila 30 içinde sonuç sağlamak için basit, çünkü hızlı grip antijen saptama testleri (RIDTs) yaygın nokta-bakım testleri gibi klinik pratikte kullanılır; Ancak, onların duyarlılıkları yaygın (% 10-80) üreticisi ve nüfus büyüklüğüne göre test edilmekte değişir ve influenza tip ve subtyPE 13, 14, 15. Doğrudan floresan deneyleri (DFA&#39;ler) RIDTs üzerinde üstün hassasiyetleri sağlar, ancak işlem süresi daha büyük (~ 3 saat) ve vasıflı teknoloji 16, 17 ile tamamlanması gerekir. Viral kültür grip teşhis için altın standart olmuştur ve her iki RIDTs ve DFAs 18 üzerinde hassasiyet geliştirdi. Ancak, grip viral kültür hasta yönetimini 19 yardımında yararını azalan tamamlamak için 2-14 d sürebilir. Son olarak, nükleik asit amplifikasyon testleri (NAAT) grip teşhisi yeni altın standart olarak kültür teknikleri yerini almıştır. NAAT birkaç saat içinde Grip taramada kullanılan büyük hassasiyeti olduğu kabul edilir. Ancak, NAAT en pahalı deneyler ve 5 gerçekleştirmek için özel ekipman ve teknoloji gerektiren <sYukarıya&gt;, 20, 21, 22, 23, 24, 25. Burada anlatılan influenza A ve B tahlil hazır ilk CLIA-feragat moleküler hızlı grip testidir. Bu deney, moleküler işaret probları ile hedef tespiti ve ardından influenzaya spesifik primerler ile izotermal amplifikasyon kullanan bir Kırpma Endonükleaz amplifikasyon reaksiyonu (yakın) kullanılarak çalışmaktadır. Bu deney, B influenza A ayıran kurmak ve bir örnek işlemek için 2 dk gerektirir ve tamamlamak için 15 dakika olmak üzere toplam gerektirir. Burada, Influenza A ve B testi için protokol mevcut. Buna ek olarak, biz örnek bir veri (viral taşıma ortamında saklanan örneklerinin arşivlenmiş nazofarengeal sürüntü (NPS) üzerine Influenza A performans ve B testi karşılaştıran VT set sağlamakBaşka solunum patojen paneli tahlil M).

<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

Grip insanlarda grip virüslerinin A ve B&#39;nin neden olduğu bulaşıcı bir solunum yolu hastalığı olan ve morbidite ve mortalite her yıl önemli miktarda neden olur. Grip A ve B tahlil birinci CLIA-feragat moleküler hızlı gribi testi kullanılabilir. Grip A ve B test moleküler işaret probları ile hedef tespiti ve ardından influenzaya spesifik primerler ile izotermal amplifikasyon kullanılarak çalışmaktadır. Burada, donmuş Grip A performans ve B tahlil, bir solunum paneli deneyi ile karşılaştırılmıştır örneklerinin viral taşıma ortamı (VTM) saklanan nazofarengeal çubukla (NPS) arşivlenmiş. Grip A ve B testinin performansı solunum paneli referans yöntemi sonuçları karşılaştırılarak değerlendirildi. toplam grip virüsü A duyarlılık% 67.5 (% 95 CI (CI), 56,6-78,5) ve özgüllük 86.9% (CI, 71,0-100) idi. İnfluenza virüsü B testi için, duyarlılık ve seçicilik% 90.2 idi (CI, 68,5-100sırasıyla) ve% 98.8 (Cl, 68,5-100),. Bu sistem tam entegre, kapalı, az yer kaplayan sistemi üzerinde çalışan herhangi bir başka mevcut moleküler tahlil ve basit, pipet serbest prosedür daha kısa bir test süresi avantajına sahiptir. Genel olarak, bu çalışmada değerlendirilen Influenza A ve B tahlil, bir nokta-bakım hızlı grip tanı testi olarak hizmet etme potansiyeline sahiptir.

Bu çalışmada, arşivlenmiş NPS örnekleri 2013 NPS örnekleri 3 ml sunuldu 15 Aralık 2012 ve 1 Mart arasında bir grip salgını sırasında Memorial Sloan-Kettering Kanser Merkezi&#39;nde (MSKCC) de grip benzeri semptomlar ile başvuran yatan hasta toplandı VTM ve çalışma süresince Influenza A, A-1, A-3, ve B olmak üzere solunum yolu virüsleri (RP), bir panel tespit moleküler testi ile rutin klinik pratikte bir parçası olarak test 3675 NPS örnekler sunuldu test için MSK...

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

Biz moleküler temelli İnfluenza A, hızlı ve B tahlil açıklar. Grip deneyi moleküler işaret probları ile hedef tespiti ve ardından influenzaya spesifik primerler ile izotermal amplifikasyon kullanılarak 15 dakika içinde her bir hedef tespit eder. Grip A ve B tahlil kullanıcı dostu ve gerçekleştirmek için en az eller-zaman gerekli.

Hızlı moleküler algılama ve Nezle Virüsleri A ve B farklılaşması

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

15.7K Görüntüleme.  Memorial Sloan Kettering Cancer Center.  Biz moleküler temelli İnfluenza A, hızlı ve B tahlil açıklar. Grip deneyi moleküler işaret probları ile hedef tespiti ve ardından influenzaya spesifik primerler ile izotermal amplifikasyon kullanılarak 15 dakika içinde her bir hedef tespit eder. Grip A ve B tahlil kullanıcı dostu ve gerçekleştirmek için en az eller-zaman gerekli. 

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.

Plazmid DNA Rekombinant Grip Virüsü Üretimi

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

İmmünoloji ve Enfeksiyon

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.

Grip için DNA Mikroarray ampliPHOX Kolorimetrik Algılama

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.

Tüm Influenza Kantitatif Analiz Basit Slot Blot Tahliller Evrensel Antikorlar kullanarak Viral Hemagglutinins ve nöraminidaz yazın

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.

Yabani Kuşlar Kuş Gribi Virüsü Hızlı Teşhis: Alanında Taşınabilir RRT-PCR ve Dondurulmuş kuru Reaktifler Kullanımı

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.

Gribi için yüksek verimlilik Tespit Yöntemi

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.

Mikroakışkan Chip İmalat ve İnfluenza Algılama Yöntemi

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

Biyomühendislik

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.

Floresans tabanlı Nöraminidaz İnhibisyonu Tahlili antivirallerin nöraminidaz inhibitörü Class Nezle Virüslerinden eğilimi değerlendirilebilir

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.

Grip A virüs enfeksiyonu bir fare modeli çalışmalarda

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.

Virüs subtypes arasında serum antikorların genişliğini ölçmek için bir Influenza antijen microarray kullanımı

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

Solunum Virüsü Tespiti için Multipleks RT-qPCR Testi

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.

Yazar Spotlight: Solunum Yolu Virüslerinin Multipleks Tespitindeki Gelişmeler 

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.

SARS-CoV-2, Influenza A/B ve MERS-CoV'nin Tespiti için Multipleks Gerçek Zamanlı RT-qPCR Testlerinin Geliştirilmesi

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