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).

ÉTICA DECLARAÇÃO: A utilização de sobras de amostras clínicas é aprovado e segue as diretrizes da Cancer Center Institutional Review Board Memorial Sloan Kettering. 1. Antes de realizar o teste NOTA: O Influenza A e B do ensaio é aprovado para espécimens de esfregaço nasofaríngeo e das zaragatoas nasofaríngeas armazenados no meio de transporte viral. As mechas são incluídos no kit e deve ser usado para um desempenho óptimo. No entanto, rayon, espuma, reuniram-se cotonetes ou swab nasal de poliéster também pode ser usado para coletar amostras de swab nasal. <ol><li> Para a coleta de uma amostra de swab nasal, insira o cotonete na narina que tem a drenagem mais visível ou que é mais congestionado. Com a rotação suave, empurre a zaragatoa na narina até encontrar resistência e rodar várias vezes contra a parede nasal antes de lentamente retirar da narina. Armazenar os cotonetes e transportá-los em frascos contendo 3 ml de meio de transporte viral (VTM). NOTA: A atenção cuidadosa para aproprcoleta de amostras iate deve ser tomado como coleta de amostra inadequada pode produzir resultados errados. NOTA: As amostras de zaragatoa deve ser testado o mais rapidamente possível após a colheita. No entanto, elas podem ser armazenadas à temperatura ambiente durante até 2 horas ou refrigerada a 2-8 ° C durante até 24 horas, se o teste não está imediatamente disponível. amostras recolhidas recentemente são ideais para o desempenho no teste ideal. O manuseio inadequado da amostra, armazenamento e / ou transporte podem produzir resultados errados. NOTA: Como medida de precaução, os indivíduos que executam este teste deve tratar todas as amostras como potencialmente infecciosas, seguindo as precauções universais ao manusear amostras. </li><li> Trazer todas as amostras à temperatura ambiente antes do teste. NOTA: Todos os componentes de ensaio são de uma única utilização e não deve ser utilizado para executar vários testes. Não misturar os componentes do kit de lotes diferentes. Não usar reagentes após a data de expiração. </li><li> Traga o receptor amostra azul para rootemperatura m antes do teste. A base de teste de laranja pode ser testada, sem a necessidade de aquecer até à temperatura ambiente. </li><li> Deixe todas as peças de teste no embrulho folha até imediatamente antes da utilização. Ligue o instrumento pressionando o botão de energia no lado do instrumento. Digite o user-ID e pressione &quot;OK&quot;. </li></ol> 2. A execução do teste <ol><li> Para iniciar o processo de teste, toque em &quot;Run Test&quot; na tela do instrumento. Digite o ID do paciente usando o teclado na tela ou scanner de código de barras e pressione &quot;OK&quot;. Abra a tampa e insira a base de teste laranja no suporte base de teste da Orange. </li><li> Confirmar que o teste correto é exibido na tela e pressione &quot;OK&quot;. Insira o receptor amostra azul no suporte do receptor amostra azul. NOTA: Não abra o receptor da amostra antes de colocar no instrumento, pois isso vai impedir que o tampão de eluição de atingir a temperatura de funcionamento correto e pode afetar o desempenho do teste. </li&gt; <li> Aguarde até que o receptor de exemplo para aquecer. Quando solicitado pelo instrumento, remover o selo de alumínio e coloque o cotonete doente a ser testado para o receptor da amostra. NOTA: Quando retirar o papel alumínio, coloque dois dedos sobre a borda do Receptor de exemplo para garantir que ele permanece no lugar. </li><li> Misture vigorosamente o cotonete no líquido durante 10 segundos, pressionando a cabeça cotonete contra a parede do receptor da amostra como você misturá-lo para ajudar a desalojar a amostra do cotonete. Pressione &quot;OK&quot; para prosseguir. Se o teste compressas armazenados em meio de transporte viral, vortex da VTM por 10 segundos, em seguida, adicione 200 mL para o receptor da amostra. </li><li> Pressionar o cartucho de transferência de branco para o receptor da amostra azul. Continue pressionando no receptor da amostra até que o indicador laranja sobe. </li><li> Levante e depois ligar o cartucho de transferência para a base de teste. Observe o indicador de laranja descer assim que a transferência cartucho está ligado corretamente. Feche a tampa e não abra até que o &quot;Teste completo&quot;mensagem aparece na tela. </li></ol> 3. Controlo de Qualidade <ol><li> Toque &#39;Run QC Test&#39; na tela inicial. Selecione o teste QC a ser executado. Confirme o tipo de teste para coincidir com a amostra QC destinado a testes tocando &#39;OK&#39; e seguir as instruções na tela para completar o teste. </li></ol> 4. Resultado Interpretação <ol><li> Após a execução do teste é completa, observar os resultados do teste apresentado no ecrã com as interpretações para a presença de Influenza A, B, subtipos desconhecidos. Para resultados inválidos repetir o teste. </li></ol> 5. Manutenção e Limpeza <ol><li> Limpar o aparelho e da área de banco circundante diariamente com 70% de etanol ou uma solução de lixívia a 10%. Pulverizar a solução de limpeza em um pano úmido, sem fiapos para limpar. Não deite as soluções diretamente no aparelho. </li></ol>

<ol>
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The authors have nothing to disclose.

Os vírus da gripe são causas mundiais de morbidade e mortalidade. diagnóstico rápido e preciso da gripe é uma das principais chaves para gerir surtos de gripe durante a temporada respiratória. Outros testes à base de antigene é rápido e fácil de realizar; no entanto, eles têm baixa sensibilidade 13. Por outro lado, os testes moleculares tradicionais têm melhorado a sensibilidade, mas requerem técnicos de laboratório mais experientes para realizar e são mais dispendiosas. O ensaio de Influenza A e B descritos no presente estudo e o protocolo é um ensaio molecular dispensada-CLIA, rápido para Influenza A e B. A tecnologia baseia-se numa amplificação isotérmica-para a detecção e diferenciação dos vírus Influenza A e B. Mostramos os resultados representativos para demonstrar o desempenho da gripe a e B no ensaio de um painel de amostras de NPS VTM arquivados, congelados foi avaliada em comparação com um outro ensaio de painel respiratório de base molecular. <p class="jove_content"&gt; As amostras que foram determinados como sendo &quot;ambígua&quot; ou &quot;untypeable&quot; pelo teste de RP teve a menor sensibilidade do ensaio de Influenza A e B. Além disso, os resultados discordantes foram observadas mais frequentemente para os espécimes com maiores valores de C T em ensaios de RP, o que sugere que a taxa de detecção mais baixo pelo ensaio foi associado com os títulos virais baixos. O ensaio teve um desempenho melhorado para a detecção de vírus da gripe B ao longo de vírus da gripe A detecção com uma sensibilidade de 90,2% e especificidade de 98,1%. Os resultados aqui são consistentes com a literatura publicada previamente 26, 27, 28. Este estudo tem duas limitações importantes. Em primeiro lugar, as amostras foram armazenadas em VTM para testar na plataforma. No entanto, no momento em que as amostras foram testadas, a plataforma não tem a aprovação da FDA para este tipo de amostra. Portanto, a diluição em VTM, pode ter reduzidoa detecção de amostras VTM de título baixo. Em segundo lugar, os espécimes que foram testados com o ensaio de Influenza A e B tinham sido armazenadas a -70 ° C durante 6 a 9 meses antes do ensaio, ao passo que o teste de RP foi feita em amostras frescas imediatamente depois de serem recebidas. É possível que o congelamento as amostras ainda mais reduzida detecção da gripe nas amostras. O ensaio de influenza A &amp; B tem alguns passos e é simples de executar. No entanto, existem duas principais etapas que devem ser concluídas de forma adequada e que os usuários podem solucionar problemas se ocorrer erros. Primeiro, quando configurar inicialmente o ensaio, o cartucho de transferência deve ser conectado à base de teste até que eles se encaixem no lugar. Se estas duas peças não são protegidos de forma adequada, a corrida vai falhar e terá de ser repetido desde o início. Em segundo lugar, o instrumento deve aquecer-se durante 2 min antes da adição da amostra. Após este tempo aquecer, os usuários têm 30 segundos para adicionar a amostra, conecte a transferência cartridge à base de teste, e feche a tampa. Se o tempo passa antes desta etapa é concluída, o instrumento irá falhar a execução e a amostra configurar terá que ser repetido desde o início. As limitações do ensaio incluem um ligeiro aumento mãos no tempo e menor sensibilidade em comparação com o painel respiratório e não subtipo de influenza A. Além disso, os únicos tipos de amostras que podem ser utilizadas com o ensaio são zaragatoas da nasofaringe ou os NPS que são armazenados em meio de transporte viral; o desempenho do ensaio para qualquer outro tipo de amostra, tais como espécimes do tracto respiratório inferior, não foi avaliada. É importante observar que todos os passos descritos neste protocolo são críticos. Quaisquer modificações ou desvio do protocolo de escrita pode resultar em menor desempenho ou do ensaio e deve ser avaliado pelo usuário. A vantagem da tecnologia é que o instrumento tem uma pequena pegada, leva apenas 15 minutos para executar e, mesmo que a tecnologiaé baseado em métodos moleculares, não requer treinamento especial ou qualificações para executar. O ensaio de Influenza A e B avaliadas neste estudo tem o potencial de servir como uma alternativa à RIDTs para o diagnóstico da gripe. O sistema tem a vantagem de um tempo total de ensaio curto, tem uma pequena pegada e é fácil de usar. Em geral, o desenho do aparelho é apropriado para testes de ponto-de-cuidados.

Infecções de vírus da gripe resultar em uma quantidade significativa de morbidade e mortalidade em cada ano 1, 2, 3. Influenza não complicada é caracterizada por sintomas respiratórios e constitucionais, tais como febre, mialgia, dor de cabeça e tosse não produtiva 4, 5. Os indivíduos mais velhos, crianças, doentes imunocomprometidos e pacientes com comorbidades subjacentes correm um risco maior de complicações graves, como pneumonia, miocardite, doença do sistema nervoso central, ou morte 6, 7. Infecção por influenza é original de outros vírus respiratórios em que a administração rápida de terapêutica antiviral dentro de 48 h do início dos sintomas pode reduzir a gravidade da doença e comprimento 8. A identificação rápida da gripe, também tem sidomostrado para reduzir o uso de antibióticos desnecessários 9, 10. Além disso, pacientes hospitalizados com infecções de gripe devem ser colocados em quartos isolados com precauções de controle de infecção apropriadas. No entanto, doenças respiratórias provocadas por vírus influenza não pode ser difícil distinguir clinicamente de gripe. Por esse motivo, os testes de diagnóstico rápidos e precisos para a gripe é muito importante para a gestão clínica do paciente. Vários ensaios estão disponíveis para a detecção e identificação dos vírus da gripe. Os testes de detecção de antígeno da gripe rápidas (RIDTs) são amplamente utilizados na prática clínica como testes point-of-care, porque eles são simples de usar e fornecer resultados dentro de 15 a 30 min 11, 12; no entanto, as suas sensibilidades variam muito (10-80%), dependendo do fabricante e da população que está sendo testado, eo tipo de gripe e subtyPE 13, 14, 15. Ensaios de fluorescência direta (DFAS) fornecem sensibilidades superiores sobre RIDTs, mas o tempo de processamento é maior (~ 3 horas) e deve ser concluída por técnicos qualificados 16, 17. A cultura viral tem sido o padrão ouro para o diagnóstico da gripe e melhorou a sensibilidade sobre ambos os RIDTs e DFAs 18. No entanto, a cultura viral influenza pode levar de 2-14 d para completar, diminuindo sua utilidade em ajudar manejo do paciente 19. Por último, os testes de amplificação de ácidos nucleicos (NAAT) substituíram as técnicas de cultura como o novo padrão ouro no diagnóstico da gripe. NASC são considerados como tendo a maior sensibilidade para a detecção de gripe em poucas horas. No entanto, NASC são os ensaios mais caros e exigem equipamentos e técnicos especializados para realizar 5 <sup&gt;, 20, 21, 22, 23, 24, 25. O ensaio de influenza A e B aqui descrito é o primeiro teste da gripe dispensada-CLIA molecular rápida que está prontamente disponível. Este ensaio trabalha utilizando uma Reacção nicking endonuclease de amplificação (NEAR) que utiliza amplificação isotérmica com iniciadores específicos de influenza, seguido de detecção do alvo com as sondas farol molecular. Este ensaio diferencia influenza A com B, requer dois minutos para configurar e processar uma amostra, e exige um total de 15 minutos para se completar. Aqui, apresentamos o protocolo para o ensaio Influenza A &amp; B. Além disso, nós fornecemos um conjunto de dados de amostra definida comparando o desempenho da Influenza A e B do ensaio de swab nasofaríngeo arquivado (NPS) amostras armazenadas em meio de transporte viral (VTM) para outro ensaio painel patógeno respiratório.

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			<td height="40" style="height:40px;width:231px;">Alere i Instrument</td>
			<td style="width:64px;">Alere</td>
			<td style="width:303px;">NAT-000 (Global), NAT-024 (US)</td>
			<td style="width:64px;"></td>
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			<td height="20" style="height:20px;">Alere i Influenza A &#38; B 24 Test Kit</td>
			<td>Alere</td>
			<td style="width:303px;">425-000 (Global), 425-024 (US)</td>
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			<td height="20" style="height:20px;">Alere i Barcode Scanner</td>
			<td>Alere</td>
			<td>EQ001001</td>
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			<td height="20" style="height:20px;">Alere Universal Printer</td>
			<td>Alere</td>
			<td style="width:303px;">55115 (Global), alereiprinter&#160;(US)</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">200 &#181;L precision pipette</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">200 &#181;L disposable pipette tips</td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;">Viral transport medium</td>
			<td>Remel</td>
			<td>M4-RT</td>
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rapid molecular detection and differentiation of influenza viruses a and b

A gripe é uma doença respiratória contagiosa causada por vírus da gripe A e B em seres humanos e faz com que uma quantidade significativa de morbilidade e mortalidade a cada ano. A Influenza A e B do ensaio foi o primeiro teste molecular gripe rápida dispensada-CLIA disponível. O teste de Influenza A e B funciona através do emprego de amplificação isotérmica com iniciadores específicos de influenza, seguido de detecção do alvo com as sondas farol molecular. Aqui, o desempenho do ensaio de Influenza A e B em congelado, arquivado esfregaço nasofaríngeo (NPS) amostras armazenadas no meio de transporte viral (VTM) foram comparados com um ensaio de painel respiratório. O desempenho do ensaio de Influenza A e B foi avaliada por comparação dos resultados com o método de referência do painel respiratório. A sensibilidade para o vírus total de influenza A foi 67,5% (CI de 95% (IC), 56,6-78,5) e a especificidade foi de 86,9% (Cl, 71,0-100). Para os testes de vírus da gripe B, a sensibilidade e a especificidade foram de 90,2% (Cl, 68,5-100) E 98,8% (Cl, 68,5-100), respectivamente. Este sistema tem a vantagem de um tempo de teste significativamente mais curto do que qualquer outro ensaio molecular actualmente disponíveis e o processo simples, livre de pipeta é executado em um sistema totalmente integrado, fechada, de pequenas dimensões. No geral, a Influenza A e B ensaio avaliada neste estudo tem o potencial de servir como um teste de diagnóstico da gripe rápida point-of-care.

Neste estudo, os espécimes NPS arquivados foram coletadas de pacientes internados com sintomas semelhantes aos da gripe no Memorial Sloan-Kettering Cancer Center (MSKCC), durante um surto de gripe entre 15 de dezembro de 2012 e 1º de março de 2013. Os espécimes NPS foram apresentados em 3 ml da VTM e testado como parte da prática clínica de rotina com um ensaio molecular que detecta um painel de vírus respiratórios (PR), incluindo a gripe a, a-1, a-3 e B. Durante o período do estudo, 3.675 espécimes NPS foram submetidas MSKCC ao laboratório clínico para testes. Dessas amostras, 45, 425, 37, e 77 deram positivo para gripe vírus A-1, A-3, A-un-subtypable (Au), e B, respectivamente, com a RP. Um total de 360 ​​amostras positivas, 40 amostras de cada um dos subtipos de gripe, e 37 UA. Além disso, 1-2 amostras negativas que chegaram ao laboratório imediatamente antes ou depois de cada um dos espécimes positivos foram escolhidos para posteriores testing com o influenza A e B do ensaio. No total, a Influenza A e B ensaio detectados 79 vírus influenza A, 37 vírus influenza B, 240 negativos e 4 resultados inválidos (falha dos controles internos) (Tabela 1). Havia 46 resultados discrepantes com 44 amostras positivas em RP somente e dois positivos sobre a Influenza A e só ensaio B. Um ensaio de gripe com base molecular terceira foi realizada em 44 amostras discordantes que detectaram 33/43 (8 vírus influenza A, Au 21, e dois vírus de influenza B) que eram positivos pelo PR e 0/1 positivo (vírus da gripe A) pela Influenza a e B do ensaio. Um acordo entre duas ou mais ensaios foi considerada uma identificação precisa. A sensibilidade global de Influenza A e B para o vírus de ensaio total de influenza A foi de 67,5% (IC de 95% (IC), 56,6-78,5). A sensibilidade para os subtipos de influenza A foi a-1, 84,6% (Cl, 58,5-100), A-<html> 3, 90,2% (IC, 62,8-100) e Au, 21,9% (CI, 19,6-24,1). A especificidade global para o vírus influenza A foi de 86,9% (Cl, 71,0-100). Para os testes de vírus da gripe B, a sensibilidade e a especificidade foram de 90,2% (Cl, 68,5-100) e 98,8% (Cl, 68,5-100), respectivamente. No geral, os valores médios de CT em espécimes com resultados discordantes foram significativamente maiores que os obtidos para resultados concordantes (16,4 ± 0,48 vs 25,7 ± 0,51; p&gt; 0,000) (Figura 1). <img alt="figura 1" src="/files/ftp_upload/54312/54312fig1.jpg" /> Figura 1: Os valores médios de CT determinado por ensaio de RP para amostras que foram positivas e negativas, respectivamente, pelo ensaio para cada subtipo de influenza. As barras de erro representam o desvio padrão. **, P &lt;0,01; ***, P &lt;0,001.1large.jpg &quot;target =&quot; _ blank &quot;&gt; Clique aqui para ver uma versão maior desta figura. <img alt="tabela 1" src="/files/ftp_upload/54312/54312table1.jpg" /> Tabela 1: Desempenho da Influenza A &amp; B na VTM espécimes comparados com os resultados de referência.

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

Descreve-se um ensaio rápido, com base molecular da gripe A e B. O ensaio detecta Influenza cada alvo dentro de 15 min, utilizando amplificação isotérmica com iniciadores específicos de influenza, seguido de detecção do alvo com as sondas farol molecular. O ensaio de Influenza A e B é de fácil utilização e necessário hands-on tempo mínimo para executar.

Rápida Detecção Molecular e diferenciação de Influenza vírus 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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

15.5K visualizações.  Memorial Sloan Kettering Cancer Center.  Descreve-se um ensaio rápido, com base molecular da gripe A e B. O ensaio detecta Influenza cada alvo dentro de 15 min, utilizando amplificação isotérmica com iniciadores específicos de influenza, seguido de detecção do alvo com as sondas farol molecular. O ensaio de Influenza A e B é de fácil utilização e necessário hands-on tempo mínimo para executar. 

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

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

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

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

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

Combinação de adesivos de fita baseada em amostragem e de fluorescência In situ Hibridação para detecção rápida de Salmonella On Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Imunologia e Infecção

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 Detecção colorimétrica em um Microarray de DNA para Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

High-throughput Detection Method for Influenza Virus

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

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

Alta capacidade método de detecção de vírus 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, ...

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

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

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

Rápida análise de aberrações cromossômicas em linfócitos B mouse por PNA-FISH

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

Detection of True IgE-expressing Mouse B Lineage Cells

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

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

Detecção de True IgE-expressando Rato B células da linhagem

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

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

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

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

A administração intranasal de recombinantes Vacinas contra Influenza em quimérico rato Modelos para estudar a imunidade mucosal

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

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

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

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

Nasal Wipes para Influenza A detecção de vírus e Isolamento de suínos

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

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

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

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

Detecção e enriquecimento da Rare células B antígeno-específica para a análise do fenótipo e função

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

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

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

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

-Fluorescência baseado Neuraminidase Ensaio de Inibição Para avaliar a susceptibilidade do vírus influenza A de neuraminidase Inibidor de Classe Antivirais

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

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

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

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

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

Medição da gripe neutralizando Antibody Responses to A(H3N2) vírus em soros humanos por Microneutralization ensaios usando as células MDCK-SIAT1

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