Os autores gostariam de agradecer ao Centro Laboratório Nacional Calicivirus for Disease Control and Prevention (CDC) para a dom espécie de alguns aspectos positivos padrão e controle de Nov, e Laboratórios da Escola de Saúde Pública da Universidade Johns Hopkins para a prestação dos reagentes.

1. As amostras de fezes <ol><li> As amostras de fezes devem ser armazenadas congeladas a preservar o RNA. Para fazer uma suspensão a 10% fecal, tomar aproximadamente 0,1 g de amostra de fezes descongeladas e completar a 1 ml com PBS. </li><li> Alíquota em 200 ul para evitar repetidos ciclos de congelamento e descongelamento. Armazenar as alíquotas a -70 ° C. </li><li> Descongelar e centrifugar alíquotas a 4.000 g durante 10 min antes da utilização na extracção. </li></ol> 2. Preparação de partículas de sílica para extracção com guanidina e Sílica <ol><li> À temperatura ambiente, suspender 30 g de dióxido de silício e completar com dH2O até 250 mL. </li><li> Após 24 horas de sedimentação, remover 215 ml do sobrenadante por sucção. Adicionar de dH2O até 250 mL, e suspender o sedimento de sílica por agitação. </li><li> Após mais 24 horas, remover o sobrenadante por sucção, e ajustar o pH da suspensão de sílica para pH 2,0, utilizando ácido clorídrico (HCl). Alíquota da suspensão de sílica em bot vidroTLEs e autoclave. </li></ol> 3. Preparação de L6 tampão para extração com Guanidina e Sílica <ol><li> À temperatura ambiente, preparar L6 tampão por dissolução de 60 g de tiocianato de guanidínio (GuSCN) e 1,3 g de Triton X-100 em 50 mL de 0,1 M cloridrato de Tris, pH 6,4, e 11 mL de uma 0,2 M EDTA, a solução de pH 8,0. </li></ol> 4. Preparação do Tampão de L2 para extração com Guanidina e Sílica <ol><li> À temperatura ambiente, preparar tampão L2 por dissolução de 180 g de tiocianato de guanidínio (GuSCN) em 150 mL de 0,1 M cloridrato de Tris, pH 6,4. </li></ol> 5. Processo de Extracção <ol><li> Em cada uma extracção NoV amostra de fezes positiva, como controlo positivo, e água tratada com DEPC, como controle negativo, deve ser incluída. </li><li> Num tubo de centrífuga de micro-misturar o seguinte; 1 mL de tampão de L6, 20 uL de partículas de sílica, e adicionar 200 uL da suspensão de extracto de 10% fecal. Vortex brevemente e deixe à temperatura ambiente por 15 min. </li><li> Centrifugar a suspensão a 6000 g durante 10 s e lavar o sedimento com 1 ml de tampão de L2 duas vezes, seguido por duas lavagens com etanol a 70% e uma vez com acetona. Seca-se o sedimento em um bloco de aquecimento a 56 ° C durante 5 min. </li><li> Hidrato o RNA no pelete de sílica através da adição de 50 uL de RNase dH 2 0. Adicionar 1 mL de RNasin, mistura invertendo o tubo e incubar a 56 ° C durante 15 min. </li><li> Centrifugar durante 3 min a toda a velocidade, numa centrífuga de mesa, e recolher 40 uL do sobrenadante contendo o RNA. </li><li> Quantificar as amostras extraídas de RNA usando Nanodrop 2000 espectrofotômetro (Thermo científico). Em seguida, armazenar a -70 ° C até à sua utilização, até 6 meses. (Nota: A melhor maneira de preservar intacto o RNA total é convertê-la em cDNA). </li></ol> 6. Extração alternativa Usando o Comercial RNA Viral RNA QIAamp Kit As instruções completas são encontradas na prov livretoided com o kit QIAGEN. Resumidamente, o procedimento é como se segue: <ol><li> Pipete 560 de Tampão AVL contendo o RNA transportador para um tubo de 1,5 mL, e adicionar 140 uL da suspensão de fezes de 10%. Mix por pulso vórtex para 15 seg. Incubar a RT durante 10 min. </li><li> Adicionar 560 uL de etanol (96-100%) para a amostra e misturar por pulso vórtex durante 15 segundos, depois brevemente centrifugar. </li><li> Aplicar 630 uL desta solução a uma coluna de spin colocado num tubo de recolha de 2 mL. Centrifugar durante 1 min a 6.000 g; então colocar a coluna de spin em um tubo limpo mL 2. </li><li> Lavar a coluna contendo RNA ligados com 500 uL de tampão de AW1 e centrifugar a 6000 g durante 1 min. Coloque em uma coluna de tubo de coleta limpo 2 mL. </li><li> Lavar a coluna com 500 uL de tampão e centrifuga-se AW2 a toda a velocidade (20.000 g ou 14.000 rpm) durante 3 min. </li><li> Coluna de spin em lugar de 1,5 mL tubo limpo micro centrífuga, adicionar 40 água tratada com DEPC-tratada uL e eluir o RNA à velocidade máxima. Repita emce para um uL 80 final de ARN eluídas. </li><li> Quantificar as amostras extraídas de RNA usando Nanodrop 2000 espectrofotômetro (Thermo científico). Em seguida, armazenar a -70 ° C até à sua utilização, até 6 meses. (Nota: A melhor maneira de preservar intacto o RNA total é convertê-la em cDNA). </li></ol> 7. Detecção de norovírus no RNA extraídos por um passo de tempo real de RT-PCR com sondas específicas TaqMan Instrumento StepOnePlus Real Time PCR Systems (Applied Biosystems). Metodologia <ol><li> Limpe e Limpe todas as superfícies de trabalho, pipetas, centrífugas e com RNase AFASTADO para eliminar qualquer contaminação RNase potencial. </li><li> Pipetar o GI e GII NoV triagem mestre mistura de acordo com a Tabela 1. Sondas Primers e Taqman estão listados na Tabela 2. </li><li> Alíquota de 10 uL de master mix adequado a cada tubo de reação. </li><li> Adicionar 5 uL de amostra desconhecida não diluídoRNA, água tratada com DEPC-tratada como controle negativo, ou GI respectivamente GII controlo positivo de RNA, aos poços de reacção correspondentes. Todas as amostras devem ser executados em duplicata. Os controlos positivos e padrões com concentração de 300 ug / uL e 500 ug / uL, respectivamente, foram fornecidas pelo Laboratório Nacional Calicivirus Centro de Controle e Prevenção de Doenças (CDC). </li><li> Centrifugar a placa de reacção em 6000 g durante 10 seg. </li><li> Na tela de propriedades do experimento; definir e selecionar um tipo de experimento para a execução. Assegure-se os reagentes TaqMan são apresentados como o tipo de reagentes, e que leitura pré-PCR e amplificação são seleccionados. </li><li> Executar 15 uL reacções usando o perfil térmico na Tabela 3. </li><li> Analisar os resultados. </li></ol> 8. Genotipagem de NoV GI e GII Usando RT-PCR convencional e Sequenciamento (Não é mencionado no vídeo, já que é um método comum e amplamente utilizado.) Instrumento. Applied Biosystems StepOne e Sistemas de Tempo StepOnePlus reais PCR com o modelo Quantitec. Metodologia <ol><li> Antes de genotipagem, o genogrupo (GI ou GII) das amostras são determinados pelo rastreio de RT-PCR acima descrito. GI NoV RNA tem de ser amplificada com a mistura mestre genotipagem GI e GII NoV o RNA com a mistura GII mestre genotipagem. </li><li> Pipetar o GI e GII NoV genotipagem mestre mistura conforme a Tabela 4. Primers GI SKF / SKR e G2 SKF / SKR estão listados na Tabela 5. </li><li> UL alíquota 45 da mistura principal apropriado para tubos de reacção de 0,2 mL. </li><li> Adicionar 5 uL de DEPC tratada com água a cada tubo de controlo negativo, e 5 uL de um pré-filtrada, Nov (GI e / ou GII) RNA positivas para o tubo de reacção correspondente. </li><li> Correr 50 uL reacções usando o perfil térmico na Tabela 6. </li><li> Enviar produtos de PCR containing, pelo menos, 100 ng / uL da região da cápside amplificado para Macrogen Companhia para a purificação e sequenciação. (9700 Grande Seneca Hwy. Rockville, MD 20850 e US $ 10 por amostra por seqüenciamento). </li></ol> 9. Os resultados representativos <img src="/files/ftp_upload/3232/3232fig1.jpg" alt="A Figura 1" /> A Figura 1 mostra resultados representativos do Taqman Um passo-RT-PCR, quando usada para RNA de ensaio extraídos a partir de amostras de fezes de crianças diarréicas. O ciclo threshold (Ct) para uma amostra positiva foi fixado em menor ou igual a 37 Ct para GI e GII 39 para Ct. O CT-valores para os controles positivos foram encontrados para ser inferior a 27 e 18 da junção ORF1-ORF2 para GI e GII, respectivamente. <a href="http://www.jove.com/files/ftp_upload/3232/3232fig1large.jpg" target="_blank">Clique aqui para ver maior figura</a> . <img src="/files/ftp_upload/3232/3232fig2.jpg" alt="A Figura 2" /> A Figura 2 </strong&gt; mostra uma comparação head-to-head de valores de Ct no método de sílica de guanidina e kit QiAmp RNA viral. Dados de ambos os testes cair muito perto da linha de igualdade (declive = 1 e intercepta = 0). Isto é confirmado pela análise de regressão eo coeficiente de correlação. Todos os controles negativos foram avaliados e considerados 0 por ambos os métodos. <table border="1"><tbody><tr><td> Mix Master </td><td> Concentração final </td><td> Volume (uL) </td></tr><tr><td> H 2 0 PCR </td><td></td><td> 2,875 </td></tr><tr><td> Quantitect RT-PCR Master Mix </td><td> 1x </td><td> 6,250 </td></tr><tr><td> RT Mix </td><td> 1x </td><td> 0,125 </td></tr><tr><td> 50 mM Cog cartilha R </td><td> 1 uM </td><td> 0,250 </td></tr><tr><td> 50 primers F mM Cog </td><td> 1mM </td><td> 0,250 </td></tr><tr><td> 10 sonda Anel mM </td><td> 1 uM </td><td> 0.125/0.250 * </td></tr><tr><td></td><td></td><td> 10,00 </td></tr></tbody></table> * UL 0,250 de cada sonda foi utilizada para o teste GI, enquanto 0,125 L de cada sonda foi utilizada para o teste GII. Tabela 1. Qiagen Quantitect master mix para triagem GI e GII (Trujillo et al., 2006) 7. <table border="1"><tbody><tr><td> Nome </td><td> Geno-grupo </td><td> Usar </td><td> Sequência (5 &#39;para 3&#39;) </td></tr><tr><td> Cog 1R </td><td> GI </td><td> Cartilha </td><td> CTT AGA CGC CAT CAT CAT TYA C </td></tr><tr><td> Cog 1F </td><td> GI </td><td> Cartilha </td><td> CGY TGG ATG CGN TTY CAT GA </td></tr><tr><td> Cog 2R </td><td> GII </td><td> Cartilha </td><td> TCG ACG TCT CCA TCA TTC ACA </td></tr><tr><td> Cog 2F </td><td> GII </td><td> Cartilha </td><td> CAR GAR BCN ATG TTY AGR TGG ATG AG </td></tr><tr><td> Anel 1A </td><td> GI </td><td> Probe </td><td> FAM-AGA TYG CGA TCY CCT GTC CA-BHQ-1 </td></tr><tr><td> 1B Anel </td><td> GI </td><td> Probe </td><td> FAM-AGA TCG CGG TCT CCT GTC CA-BHQ-1 </td></tr><tr><td> Ring2-TP </td><td> GII </td><td> Probe </td><td> FAM-TGG GGC GAG GAT CGC AAT CT-BHQ-1 </td></tr></tbody></table> Tabela 2. Primer e oligonucleótidos sonda utilizada em tempo real RT-PCR quantitativo para genogrupos I, II (Kageyama et al.,2003) 8. <table border="1"><tbody><tr><td> Passo </td><td> Temp (° C) </td><td> Tempo (min) </td><td></td></tr><tr><td> 1 </td><td> 50 </td><td> 30:00 </td><td> síntese de cDNA </td></tr><tr><td> 2 </td><td> 95 </td><td> 15:00 </td><td> HotStart activação polimerase Taq </td></tr><tr><td> 3 </td><td> 95 </td><td> 00:15 </td><td></td></tr><tr><td></td><td> 60 </td><td> 01:00 </td><td> Ciclos 45x </td></tr></tbody></table> Tabela3. Perfil térmico para uma etapa Taqman em tempo real RT-PCR (Trujillo et al., 2006) 7. <table border="1"><tbody><tr><td> Mix Master </td><td> Concentração final </td><td> Volume (uL) </td></tr><tr><td> H 2 0 PCR </td><td></td><td> 29,50 </td></tr><tr><td> 5X Qiagen RT-PCR Tampão </td><td> 1x </td><td> 10,00 </td></tr><tr><td> 10 mM mistura de dNTP </td><td> 0,4 mM </td><td> 2,00 </td></tr><tr><td> Mix enzima </td><td></td><td> 2,00 </td></tr><tr><td> 40 RNasin U / uL </td><td> 20 U / uL </td><td> 0,50 </td></tr><tr><td> 10 uM SKF </td><td> 0,1 mM </td><td> 0,50 </td></tr><tr><td> 10 SKR mM </td><td> 0,1 mM </td><td> 0,50 </td></tr></tbody><tbody><tr><td></td><td></td><td> 45,00 </td></tr></tbody></table> Tabela 4. Qiagen One-Step RT-PCR master mix para genotipagem GI e GII. <table border="1"><tbody><tr><td> Nome </td><td> Geno-grupo </td><td> Usar </td><td> Sequência (5 &#39;para 3&#39;) </td></tr><tr><td> G1SKF </td><td> GI </td><td> Cartilha </td><td> 5&#39;-CTG CCC GAA TTY GTA AAT GA - 3 </td></tr><tr><td> G1SKR </td><td> GI </td><td> Cartilha </td><td> 5&#39;-CCA CAR ACC CCA TTR TAC A - &#39;3 </td></tr><tr><td> G2SKF </td><td> GII </td><td> Cartilha </td><td> 5&#39;-GGG CNT AGG GCG ATC GCA A - 3 </td></tr><tr><td> G2SKR</td><td> GII </td><td> Cartilha </td><td> 5&#39;-GCA CCR CCN TRH CCR TTR TA CAT-3 </td></tr></tbody></table> Primers Tabela 5. Utilizados para a amplificação da região C da Região da Cápside (Kojima et ai., 2002). 5 <table border="1"><tbody><tr><td> Passo </td><td> Temp (° C) </td><td> Tempo (min) </td><td></td></tr><tr><td> 1 </td><td> 60 </td><td> 30:00 </td><td> síntese de cDNA </td></tr><tr><td> 2 </td><td> 96 </td><td> 15:00 </td><td> HotStart activação polimerase Taq </td></tr><tr><td> 3 </td><td> 94 </td><td> 00:030 </td><td></td></tr><tr><td></td><td> 52 </td><td> 01:00 </td><td> Ciclos 40X </td></tr><tr><td></td><td> 72 </td><td> 00:30 </td><td></td></tr><tr><td> 4 </td><td> 72 </td><td> 10:00 </td><td> Recozimento </td></tr></tbody></table> Tabela 6. Perfil térmico para Taqman One-Step RT-PCR.

<ol>
	<li>Medici, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+epidemiology+of+Norovirus+infections+in+sporadic+cases+of+viral+gastroenteritis+among+children+in+Northern+Italy.">Molecular epidemiology of Norovirus infections in sporadic cases of viral gastroenteritis among children in Northern Italy.</a> L. Medical Virology. 78, (2006).</li><li>Vidal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+recombinant+Norovirus+causing+outbreaks+of+gastroenteritis+in+Santiago,+Chile.">Novel recombinant Norovirus causing outbreaks of gastroenteritis in Santiago, Chile.</a> J. Clinica Microbiology. 4, (2006).</li><li>Xavier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+caliciviruses+associated+with+acute+infantile+gastroenteritis+in+Salvador,+an+urban+center+in+Northeast+Brazil.">Detection of caliciviruses associated with acute infantile gastroenteritis in Salvador, an urban center in Northeast Brazil.</a> Braz. J. Med. Biol. Res. 42, (2009).</li><li>Boom, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+simple+method+for+purification+of+nucleic+acids.">Rapid and simple method for purification of nucleic acids.</a> J. Clin. Microbiol. 28, 495-503 (1990).</li><li>Kojima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genogroup-specific+PCR+primers+for+detection+of+Norwalk-like+viruses.">Genogroup-specific PCR primers for detection of Norwalk-like viruses.</a> J. Virol. Methods. 100, 107-114 (2002).</li><li>Mattison, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+comparison+of+two+norovirus+ORF2-based+genotyping+protocols.">Multicenter comparison of two norovirus ORF2-based genotyping protocols.</a> J. Clin. Microbiol. 47, 3927-3932 (2009).</li><li>Trujillo, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+TaqMan+real-time+reverse+transcription-PCR+for+rapid+detection,+quantification,+and+typing+of+norovirus.">Use of TaqMan real-time reverse transcription-PCR for rapid detection, quantification, and typing of norovirus.</a> J. Clin. Microbiol. 44, 1405-1412 (2006).</li><li>Kageyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+broadly+reactive+and+highly+sensitive+assay+for+Norwalk-like+viruses+on+real-time+quantitative+RT-PCR.">A broadly reactive and highly sensitive assay for Norwalk-like viruses on real-time quantitative RT-PCR.</a> J. Clin. Microbiol. 41, 1548-1557 (2003).</li></ol>

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Usando o económico in-house método para isolar o ácido nucleico a partir de amostras de fezes, obtém-se resultados iguais como com o comercial QIAmp de RNA viral kit de Qiagen, e em conjunto com o TaqMan RT-PCR desenvolvido no nosso laboratório que pode detectar uma ampla gama de NoV genótipos pertencentes ao GI e GII genogrupo. Uma publicação recente da região C protocolo relataram taxas de genotipagem de 78% 6. Uma vez que a diversidade das cepas contemporâneas Nov tem vindo a aumentar nos anos an...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Isotiocianato de guanidina </td><td> Sigma-Adrich </td><td> G9277 </td><td></td></tr><tr><td> Tris-HCl </td><td> Sigma-Aldrich </td><td> T5941 </td><td></td></tr><tr><td> EDTA </td><td> Sigma-Aldrich </td><td> E5134 </td><td></td></tr><tr><td> Sílica </td><td> Sigma-Aldrich </td><td> S5631 </td><td></td></tr><tr><td> Triton X 100 </td><td> BDH Chemicals </td><td> 14530 </td><td></td></tr><tr><td> Dietilpirocarbonato </td><td> Sigma-Aldrich </td><td> D-5758 </td><td></td></tr><tr><td> QIAamp viral RNA Mini Kit (250) </td><td> QIAGEN </td><td> 52906 </td><td></td></tr><tr><td> QuantiTec Probe RT-PCR kit (200) </td><td> QIAGEN </td><td> 204443 </td><td></td></tr><tr><td> Qiagen Um passo de RT-PCR Kit (200) </td><td> QIAGEN </td><td> 210212 </td><td></td></tr><tr><td> Rnase Inibidor 2000 unidades </td><td> A.Biosystems </td><td> N808-0119 </td><td> 2000 unids / frasco </td></tr><tr><td> Antiaderentes Rnae-free tubos de microcentrífuga </td><td> Ambion </td><td> AM12450 </td><td></td></tr><tr><td> UltraPure Agarose 1000 </td><td> Invitrogen </td><td> 16550-100 </td><td></td></tr></tbody></table>

detection and genogrouping of noroviruses from children's stools by taqman one-step rt-pcr

Os norovírus (NoVs) são a principal causa de surtos de gastroenterite aguda esporádica em todo o mundo em seres humanos de todas as idades. Eles são importante causa de hospitalizações em crianças com impacto na saúde pública semelhante à de Rotavirus. NoVs são vírus de RNA de grande diversidade genética e há uma aparência contínua de novas estirpes. Cinco genogrupos são reconhecidos; GI e GII com seus genótipos e subtipos muitos sendo o mais importante para a infecção humana. No entanto, o diagnóstico de dois genótipos continua a ser problemática, retardando o diagnóstico e tratamento. 1, 2, 3 Para extração de RNA a partir de amostras de fezes, o método mais comumente usado é o QIAmp kit RNA Viral comercial da Qiagen. Este método combina as propriedades de ligação de uma membrana de gel de sílica, buffers que as RNases de controlo e proporcionam ligação óptimas de ARN para a coluna, juntamente com a velocidade de MicroSpin. Este método é simples, rápido e confiável e é carrIED em alguns passos que são detalhados na descrição fornecida pelo fabricante. O norovírus é apenas a segunda rotavírus como a causa mais comum de diarréia. Diagnóstico norovírus devem estar disponíveis em todos os estudos sobre a patogênese da diarréia, bem como em surtos ou casos de diarréia individuais. Actualmente no entanto diagnóstico norovírus é restrita a poucos centros devido à falta de métodos simples de diagnóstico. Este diagnóstico e tratamento atrasos 1, 2, 3. Além disso, devido aos custos de transporte e regulamentado de buffers corrosivos dentro e entre países o uso desses kits manufaturados coloca problemas logísticos. Como resultado, neste protocolo descrevemos uma alternativa, económica, método in-house que se baseia na lança inicial et al. Método 4 que utiliza as propriedades de ligação de ácidos nucleicos de partículas de sílica, juntamente com as propriedades anti-nuclease de tiocianato de guanidínio . <p class=&quot;Jove_content&quot;&gt; Para a detecção e genogrouping (GI e GII) de isolados NoVs de amostras de fezes, vários protocolos de RT-PCR utilizando diferentes alvos têm sido desenvolvidos. O consenso é que uma RT-PCR utilizando TaqMan química seria a melhor técnica molecular para o diagnóstico, porque combina alta sensibilidade, especificidade e reprodutibilidade com alto rendimento e facilidade de utilização. Descrevemos aqui um ensaio de segmentação da estrutura de leitura aberta 1 (ORF1) região de junção-ORF2; a região mais conservada do genoma NoV e, portanto, mais adequado para o diagnóstico. Para a análise genética mais uma RT-PCR convencional que tem como alvo o altamente variável N-terminal a partir de casca-a principal proteína da cápside (Região C) utilizando iniciadores originalmente descritos por Kojima et ai. 5 é detalhado. A sequenciação do produto de PCR a partir do PCR convencional permite a diferenciação de genótipos pertencentes ao GI e genogrupos GII.

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Detection and Genogrouping of Noroviruses from Children&#039;s Stools By Taqman One-step RT-PCR

Um Um passo-ensaio de RT-PCR para a detecção e identificação genogrupo de isolados Norovirus a partir de fezes de crianças, que utiliza iniciadores e sondas de TaqMan específicos para o quadro de leitura aberta 1 (ORF1)-ORF2 região de junção, a região mais conservada do genoma norovírus é descrito. A não-comercial, custo-efetivo método de extração de RNA é detalhado.

Detecção e Genogrouping de norovírus a partir de fezes das crianças, Taqman Um passo-RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

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Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

17.2K visualizações.  Universidad Peruana Cayetano Heredia. Um Um passo-ensaio de RT-PCR para a detecção e identificação genogrupo de isolados Norovirus a partir de fezes de crianças, que utiliza iniciadores e sondas de TaqMan específicos para o quadro de leitura aberta 1 (ORF1)-ORF2 região de junção, a região mais conservada do genoma norovírus é descrito. A não-comercial, custo-efetivo método de extração de RNA é detalhado.

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MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

Detecção MicroRNA em tumores de próstata por Quantitative PCR em tempo real (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Medicina

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Luminescência de Cerenkov Imaging (CLI) para o Monitoramento Terapia de Câncer

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Implantação de Veia Cava Inferior interposição de enxerto no Rato Modelo

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Isolamento e Imortalidade de linhas celulares derivadas do paciente a partir de biópsia muscular para a doença Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

Estabelecimento de epiteliais humanas Enteroids e Colonoids de tecido inteiro e biópsia

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

Infusão do ducto pancreático: um método eficaz e seletivo de medicamentos e entrega viral

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

Isolamento dos hepatócitos primários de rato para análise de síntese de proteína nascente pelo método de rotulagem não-radioativo L-azidohomoalanine

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Handheld Metal Detector Screening for Metallic Foreign Body Ingestion in Children

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a handheld metal detector to detect ingested metallic foreign bodies in children. We propose that by introducing handheld metal detector screening early in the triage process of children with high suspicion of metallic foreign body ingestion, the number of radiographs being ordered to localize the metallic foreign body can be reduced in this radio-sensitive population. The study protocol requires the screening of the participants for history of foreign body ingestion and exclusion of patients with respiratory distress or metallic implants. The patient changes to hospital gown and items that could contain metal like eyeglasses, earrings, pendants, and ornaments are removed. The patient is positioned in the center of the room away from other metallic interferences. The working status of the handheld metal detector is first confirmed by eliciting a positive audio-visual signal. Then the screening is done in an erect position with head in extension to expose the neck, from the level of the chin to the level of the hip joint, to cover the anatomical areas from neck to pelvis in a zig-zag manner both anteriorly and posteriorly. A positive audio-visual signal is carefully noted during the scanning for the presence of metallic foreign body. Relevant radiographs are ordered as per the area detected on the metal detector screening. The handheld metal detector was able to precisely identify all the coins among the ingested metallic foreign bodies in our study. The handheld metal detector could not consistently detect non-coin metallic foreign bodies. This protocol demonstrates the accuracy of handheld metal detector in the identification and localization of coins and coin like metallic foreign bodies.

Here, we present a protocol to use the handheld metal detector, to screen for the presence of ingested metallic foreign bodies in children who present to the pediatric emergency medicine department with a history of foreign body ingestion.

Triagem de Detector de metais portátil para ingestão de corpo estranho metálico em crianças

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

Detecção baseada em plaquetas de óxido nítrico no sangue, medindo-se a fosforilação VASP

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Assessment and Characterization of Hyaloid Vessels in Mice

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed regression of hyaloid vessels can be seen in diseases such as persistent hyperplastic primary vitreous (PHPV), leading to an obstructed light path and impaired visual function. Understanding the mechanisms underlying the hyaloid vessel regression may lead to new molecular insights into the vascular regression process and potential new ways to manage diseases with persistent hyaloid vessels. Here we describe the procedures for imaging hyaloid in live mice with optical coherence tomography (OCT) and fundus fluorescein angiography (FFA) and a detailed technical protocol of isolating and flat-mounting hyaloid ex vivo for quantitative analysis. Low-density lipoprotein receptor-related protein 5 (LRP5) knockout mice were used as an experimental model of persistent hyaloid vessels, to illustrate the techniques. Together, these techniques may facilitate a thorough assessment of hyaloid vessels as an experimental model of vascular regression and studies on the mechanism of persistent hyaloid vessels.

This protocol describes both in vivo and ex vivo methods to fully visualize and characterize hyaloid vessels, a model of vascular regression in mouse eyes, using optical coherence tomography and fundus fluorescein angiography for the live imaging and ex vivo isolation and subsequent flat mount of hyaloid for quantitative analysis.

Avaliação e caracterização de vasos Hialóides em camundongos

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed ...