Los autores desean agradecer al Centro de Calicivirus Laboratorio Nacional para el Control y Prevención de Enfermedades (CDC) para el tipo de regalo algunos aspectos positivos estándar y el control de Nov, y los Laboratorios de la Escuela de Salud Pública de Johns Hopkins para la prestación de los reactivos.

1. Las muestras de heces <ol><li> Las muestras de heces deben conservarse congelados para preservar el ARN. Para hacer una suspensión al 10% fecal, tome aproximadamente 0,1 g de muestra de heces descongelado y completar a 1 ml con PBS. </li><li> Dividir en partes alícuotas en 200 l para evitar los ciclos de congelación y descongelación. Almacene las alícuotas a -70 ° C. </li><li> Descongele y centrifugue alícuotas a 4.000 g durante 10 minutos antes de su uso en la extracción. </li></ol> 2. Preparación de partículas de sílice para la extracción con guanidina y sílice <ol><li> A temperatura ambiente, suspender 30 g de dióxido de silicio y completar con dH 2 O hasta 250 ml. </li><li> Después de 24 horas de sedimentación, eliminar 215 ml del sobrenadante por succión. Añadir DH 2 O hasta 250 ml, y la suspensión de la sílice precipitado por agitación. </li><li> Después de otras 24 horas, separar el sobrenadante por succión, y ajustar el pH de la suspensión de sílice a un pH de 2,0, utilizando ácido clorhídrico (HCl). Alícuota de la suspensión de sílice en el bot de vidrioTLEs y autoclave. </li></ol> 3. Preparación de la L6 de búfer de extracción con guanidina y sílice <ol><li> A temperatura ambiente, preparar L6 tampón disolviendo 60 g de tiocianato de guanidinio (GuSCN) y 1,3 g de Triton X-100 en 50 ml de 0,1 M de hidrocloruro de Tris, pH 6,4, y 11 ml de una EDTA 0,2 M, pH 8,0 solución. </li></ol> 4. Preparación del tampón de L2 para extracción con guanidina y sílice <ol><li> A temperatura ambiente, preparar tampón L2 disolviendo 180 g de tiocianato de guanidinio (GuSCN) en 150 ml de 0,1 M de hidrocloruro de Tris, pH 6,4. </li></ol> 5. Procedimiento de extracción <ol><li> En cada extracción de una muestra de heces positivo NoV, como control positivo, y agua DEPC tratados, como control negativo, se debe incluir. </li><li> En un tubo de centrífuga de micro-mezclar los siguientes; 1 ml de tampón L6, 20 l de partículas de sílice, y añadir 200 l de la suspensión extracto 10% fecal. Vortex brevemente y dejar a temperatura ambiente durante 15 millasn. </li><li> Centrifugar la suspensión a 6000 g durante 10 s, y lavar el precipitado con 1 ml de tampón L2 dos veces, seguido por dos lavados con etanol al 70% y una vez con acetona. Secar el precipitado en un bloque de calentamiento en seco a 56 ° C durante 5 min. </li><li> Hidratación el ARN en el sílice precipitado mediante la adición de 50 l de RNasa libre de dH 2 0. Añadir 1 l de RNasin, mezclar invirtiendo el tubo y se incuba a 56 ° C durante 15 min. </li><li> Centrifugar durante 3 minutos a plena velocidad, en una centrífuga de mesa, y recoger 40 l del sobrenadante que contiene el ARN. </li><li> Cuantificar los extrajeron muestras de ARN utilizando Nanodrop 2000 espectrofotómetro (Thermo Scientific). A continuación, almacenar a -70 ° C hasta su uso, hasta 6 meses. (Nota: Una mejor manera de preservar el total de ARN intacto es convertirlo en cDNA). </li></ol> 6. La extracción comercial alternativo mediante el ARN viral kit QIAamp ARN Las instrucciones completas se encuentran en el folleto provIDed con el kit de QIAGEN. Brevemente el procedimiento es como sigue: <ol><li> L Pipetee 560 de tampón AVL que contiene el ARN portadoras en un tubo de 1,5 ml, y añadir 140 l de la suspensión de heces 10%. Mezclar por vortex pulso durante 15 segundos. Incubar a temperatura ambiente durante 10 min. </li><li> Añadir 560 l de etanol (96-100%) a la muestra y mezclar por vortex pulso durante 15 segundos, centrifugar brevemente. </li><li> Aplicar 630 l de esta solución a una columna de centrifugación coloca en un tubo de recogida de 2 ml. Centrifugar durante 1 min a 6.000 g, y luego colocar la columna de centrifugación en un tubo limpio ml 2. </li><li> Lavar la columna que contiene ARN enlazados con 500 l de buffer AW1 y se centrifuga a 6.000 g durante 1 min. Coloque la columna en un tubo de recogida limpio de 2 ml. </li><li> Lavar la columna con 500 l de tampón AW2 y centrifugar a toda velocidad (20.000 g ó 14.000 rpm) durante 3 min. </li><li> La columna de giro en un lugar limpio de 1,5 ml de micro-tubo de centrífuga, añadir 40 l agua tratada con DEPC y eluir el RNA a toda velocidad. Repita el procedimiento ence para una final de 80 L de ARN eluidos. </li><li> Cuantificar los extrajeron muestras de ARN utilizando Nanodrop 2000 espectrofotómetro (Thermo Scientific). A continuación, almacenar a -70 ° C hasta su uso, hasta 6 meses. (Nota: Una mejor manera de preservar el total de ARN intacto es convertirlo en cDNA). </li></ol> 7. La detección de norovirus en el ARN extraído por el tiempo de un paso real RT-PCR con sondas específicas de Taqman Instrumento StepOnePlus PCR en Tiempo Real de Sistemas (Applied Biosystems). Metodología <ol><li> Limpiar y limpiar todas las superficies de trabajo, pipetas y centrífugas con RNasa LEJOS para eliminar cualquier potencial de contaminación RNasa. </li><li> Pipetear la GI y GII NoV detección mezclas maestras de acuerdo a la Tabla 1. Primers y sondas TaqMan están listados en la Tabla 2. </li><li> Alícuota de 10 l de mezcla maestra apropiada a cada tubo de reacción. </li><li> Añadir 5 ml de la muestra problema sin diluirARN, agua tratada con DEPC como control negativo, o IG, respectivamente, GII positivo ARN de control, a los pocillos de reacción correspondientes. Todas las muestras deben procesarse por duplicado. Los controles positivos y las normas de concentración de 300 mg / l y 500 mg / l, respectivamente, fueron proporcionados por Nacional Calicivirus Laboratorio Centro para el Control y Prevención de Enfermedades (CDC). </li><li> Centrifugar la placa de reacción a 6.000 g durante 10 seg. </li><li> En la pantalla del experimento de propiedades, definir y seleccionar un tipo de experimento para la carrera. Asegúrese de que los reactivos TaqMan se muestran como el tipo de reactivos, y que decía: pre-PCR y la amplificación se seleccionan. </li><li> Ejecutar 15 l reacciones utilizando el perfil térmico en la Tabla 3. </li><li> Analizar los resultados. </li></ol> 8. La genotipificación del NoV GI y GII Usando convencional de RT-PCR y secuenciación (No se menciona en este video, ya que es un método común y ampliamente utilizado.) Instrumento. Applied Biosystems StepOne y StepOnePlus en tiempo real de sistemas de PCR con la plantilla Quantitec. Metodología <ol><li> Antes de genotipado, el genogrupo (GI o GII) de las muestras se determinó por la detección de RT-PCR se ha descrito anteriormente. GI NoV ARN tiene que ser amplificado con la mezcla maestra genotipo GI y el GII NoV ARN con la mezcla de determinación del genotipo GII maestro. </li><li> Pipetear la GI y GII NoV genotipo maestro de las mezclas de acuerdo a la Tabla 4. Primers GI SKF / SKR y G2 SKF / SKR se enumeran en la Tabla 5. </li><li> Alícuota de 45 l de la mezcla maestra adecuada para tubos de 0,2 mL de reacción. </li><li> Añadir 5 l de agua tratada con DEPC a cada tubo de control negativo, y 5 l de pre-seleccionados, nov (GI y / o GII) ARN positivos en el tubo de reacción correspondiente. </li><li> Ejecutar 50 reacciones l utilizando el perfil térmico en la Tabla 6. </li><li> Enviar PCR productos conteNing al menos 100 ng / l de la región amplificada a cápsida Macrogen Compañía para la purificación y secuenciación. (9700 Gran Séneca carretera. Rockville, MD 20850 y $ 10 por muestra por secuenciación). </li></ol> 9. Los resultados representativos <img src="/files/ftp_upload/3232/3232fig1.jpg" alt="Figura 1" /> La figura 1 muestra los resultados representativos de la TaqMan One-Step RT-PCR cuando se utiliza para el ARN de ensayo extraídas de muestras de heces diarreicas de los niños. El ciclo umbral (Ct) de una muestra positiva se fijó en menor o igual a 37 Ct para el GI y GII 39 para Ct. El CT-valores para los controles positivos se encontraron en menos de 27 y 18 de la unión ORF1-ORF2 para el GI y GII, respectivamente. <a href="http://www.jove.com/files/ftp_upload/3232/3232fig1large.jpg" target="_blank">Haga clic aquí para ver más grande la figura</a> . <img src="/files/ftp_upload/3232/3232fig2.jpg" alt="Figura 2" /> Figura 2 </strong&gt; muestra una comparación cabeza a cabeza de los valores de Ct de la sílice en el método de guanidinio y el kit de QIAmp ARN viral. Los datos de ambas pruebas caer muy cerca de la línea de igualdad (pendiente = 1 y la intersección = 0). Esto se ve confirmado por el análisis de regresión y el coeficiente de correlación. Todos los controles negativos se evaluaron y se encontró que 0 por ambos métodos. <table border="1"><tbody><tr><td> Master Mix </td><td> Concentración final </td><td> Volumen (l) </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 micras Cog R imprimación </td><td> 1 mM </td><td> 0,250 </td></tr><tr><td> 50 micras de imprimación Cog F </td><td> 1mM </td><td> 0,250 </td></tr><tr><td> 10 mM de la sonda del anillo </td><td> 1 mM </td><td> 0.125/0.250 * </td></tr><tr><td></td><td></td><td> 10,00 </td></tr></tbody></table> * 0,250 l de cada sonda se utilizó para las pruebas de IG, mientras que 0,125 l de cada sonda se utilizó para las pruebas de la GII. Tabla 1. Qiagen Quantitect mezcla maestra para la investigación GI y GII (Trujillo et al., 2006). 7 <table border="1"><tbody><tr><td> Nombre </td><td> Geno-grupo </td><td> Utilizar </td><td> Secuencia (5 &#39;a 3&#39;) </td></tr><tr><td> Cog 1R </td><td> GI </td><td> Cartilla </td><td> CTT AGA CGC CAT CAT CAT TYA C </td></tr><tr><td> Cog 1F </td><td> GI </td><td> Cartilla </td><td> CGY TGG ATG CGN TTY CAT GA </td></tr><tr><td> Cog 2R </td><td> GII </td><td> Cartilla </td><td> TCG ACG CCA TCA TCT ACA TTC </td></tr><tr><td> Cog 2F </td><td> GII </td><td> Cartilla </td><td> CAR GAR BCN ATG TTY AGR TGG ATG AG </td></tr><tr><td> Anillo de 1A </td><td> GI </td><td> Sonda </td><td> FAM-AGA TYG CGA TCY CCT GTC CA-BHQ-1 </td></tr><tr><td> Anillo 1B </td><td> GI </td><td> Sonda </td><td> FAM-AGA TCG CGG CCT TCT GTC CA-BHQ-1 </td></tr><tr><td> RING2-TP </td><td> GII </td><td> Sonda </td><td> FAM-TGG GAG GAT GGC CGC AAT CT-BHQ-1 </td></tr></tbody></table> Tabla 2. Primer y oligonucleótidos sonda utilizada para la cuantitativa en tiempo real RT-PCR para genogrupos I, II (Kageyama et al.2003) 8. <table border="1"><tbody><tr><td> Paso </td><td> Temperatura (° C) </td><td> Tiempo (min) </td><td></td></tr><tr><td> 1 </td><td> 50 </td><td> 30:00 </td><td> síntesis de ADNc </td></tr><tr><td> 2 </td><td> 95 </td><td> 15:00 </td><td> HotStart Taq activación de la polimerasa </td></tr><tr><td> 3 </td><td> 95 </td><td> Doce y quince </td><td></td></tr><tr><td></td><td> 60 </td><td> 01:00 </td><td> Ciclos de 45X </td></tr></tbody></table> Mesa3. El perfil térmico de un solo paso Taqman en tiempo real RT-PCR (Trujillo et al., 2006). 7 <table border="1"><tbody><tr><td> Master Mix </td><td> Concentración final </td><td> Volumen (l) </td></tr><tr><td> H 2 0 PCR </td><td></td><td> 29,50 </td></tr><tr><td> 5X Qiagen RT-PCR Buffer </td><td> 1x </td><td> 10,00 </td></tr><tr><td> 10 mM dNTP Mix </td><td> 0,4 mM </td><td> 2,00 </td></tr><tr><td> Mezcla de enzimas </td><td></td><td> 2,00 </td></tr><tr><td> 40 U / l ARNsin </td><td> 20 U / l </td><td> 0,50 </td></tr><tr><td> 10 mM de SKF </td><td> 0.1 mM </td><td> 0,50 </td></tr><tr><td> 10 mM SKR </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> Tabla 4. Qiagen One-Step RT-PCR Master Mix para la determinación del genotipo GI y GII. <table border="1"><tbody><tr><td> Nombre </td><td> Geno-grupo </td><td> Utilizar </td><td> Secuencia (5 &#39;a 3&#39;) </td></tr><tr><td> G1SKF </td><td> GI </td><td> Cartilla </td><td> 5&#39;-CTG CCC GAA TTY GTA AAT GA - 3 </td></tr><tr><td> G1SKR </td><td> GI </td><td> Cartilla </td><td> 5&#39;-CCA CAC CAR CCA TTR Un TAC - &#39;3 </td></tr><tr><td> G2SKF </td><td> GII </td><td> Cartilla </td><td> 5&#39;-GGG AGG CNT GCG ATC ACG A - 3 </td></tr><tr><td> G2SKR</td><td> GII </td><td> Cartilla </td><td> 5&#39;-CCR CCN GCA TRH CCR TTR TA CAT-3 </td></tr></tbody></table> Los cebadores Tabla 5. Utilizado para la amplificación de la Región C de la región de la cápside (Kojima et al., 2002). 5 <table border="1"><tbody><tr><td> Paso </td><td> Temperatura (° C) </td><td> Tiempo (min) </td><td></td></tr><tr><td> 1 </td><td> 60 </td><td> 30:00 </td><td> síntesis de ADNc </td></tr><tr><td> 2 </td><td> 96 </td><td> 15:00 </td><td> HotStart Taq activación de la polimerasa </td></tr><tr><td> 3 </td><td> 94 </td><td> 00:30 </td><td></td></tr><tr><td></td><td> 52 </td><td> 01:00 </td><td> Ciclos de 40X </td></tr><tr><td></td><td> 72 </td><td> Doce y media </td><td></td></tr><tr><td> 4 </td><td> 72 </td><td> 10:00 </td><td> Recocido </td></tr></tbody></table> Tabla 6. El 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>

No hay conflictos de interés declarado.

Uso de la económica en casa-método para aislar ácido nucleico a partir de muestras de heces, se obtiene resultados iguales como con el kit comercial QIAmp viral de RNA de Qiagen, y junto con la TaqMan RT-PCR desarrollado en nuestro laboratorio que puede detectar una amplia gama de NoV genotipos pertenecientes al GI y GII genogrupo. Una publicación reciente del protocolo de la región C informaron las tasas de genotipado de 78% 6. Desde la diversidad de las cepas contemporáneas NoV ha ido en aumento duran...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Guanidina isotiocianato </td><td> Sigma-Aldrich </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> Sílice </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 sonda RT-PCR kit (200) </td><td> QIAGEN </td><td> 204443 </td><td></td></tr><tr><td> Qiagen One Step RT-PCR kit (200) </td><td> QIAGEN </td><td> 210212 </td><td></td></tr><tr><td> Inhibidor de RNasa de 2000 unidades </td><td> A.Biosystems </td><td> N808-0119 </td><td> 2000 unids / vial </td></tr><tr><td> Antiadherente ERNA sin tubos de microcentrífuga </td><td> Ambion </td><td> AM12450 </td><td></td></tr><tr><td> UltraPure agarosa 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

Los norovirus (NOV) son la principal causa de brotes de gastroenteritis aguda esporádica en todo el mundo en los seres humanos de todas las edades. Ellos son causa importante de hospitalización en niños con un impacto en la salud pública similar a la del rotavirus. NOV son virus de ARN de una gran diversidad genética y hay una continua aparición de nuevas cepas. Cinco genogrupos son reconocidos; GI y GII, con sus muchos genotipos y subtipos, siendo los más importantes para la infección humana. Sin embargo, el diagnóstico de estas dos genotipos sigue siendo problemática, retrasando el diagnóstico y tratamiento. 1, 2, 3 Para la extracción de RNA de muestras de heces, el método más comúnmente utilizado es el kit de QIAmp ARN viral comercial de Qiagen. Este método combina las propiedades de unión de una membrana de gel de sílice, tampones que RNasas de control y proporcionan unión óptimos de los ARN a la columna junto con la velocidad de MicroSpin. Este método es sencillo, rápido y fiable y es carrIED en unos pocos pasos que se detallan en la descripción proporcionada por el fabricante. El Norovirus es sólo superada por rotavirus es la causa más común de diarrea. Diagnóstico de norovirus deben estar disponibles en todos los estudios sobre la patogenia de la diarrea, así como en los brotes de diarrea o de casos individuales. En la actualidad, sin embargo, el diagnóstico de norovirus se limita a unos pocos centros, debido a la falta de métodos sencillos de diagnóstico. Este retraso diagnóstico y el tratamiento 1, 2, 3. Además, debido a los costos de transporte y regulación de los buffers corrosivos dentro y entre los países el uso de estos equipos fabricados plantea problemas logísticos. Como resultado, en este protocolo se describe una alternativa, económica, en casa método que se basa en el original de la pluma y col. Método 4, que utiliza las propiedades de unión de ácidos nucleicos de partículas de sílice junto con las propiedades anti-nucleasa de tiocianato de guanidinio . <p class=&quot;Jove_content&quot;&gt; Para la detección y genogrouping (GI y GII) de los aislados de muestras de heces NOV, varios protocolos de RT-PCR utilizando los objetivos se han desarrollado diferentes. El consenso es que una RT-PCR utilizando TaqMan química sería la mejor técnica molecular para el diagnóstico, ya que combina una alta sensibilidad, especificidad y reproducibilidad con un alto rendimiento y facilidad de uso. Aquí se describe un ensayo de orientación del marco de lectura abierto 1 (ORF1)-ORF2 región de unión; la región más conservada del genoma NoV y por lo tanto más adecuado para el diagnóstico. Para su posterior análisis genético una RT-PCR convencional que se dirige a la muy variable N-terminal de concha de la principal proteína de la cápside (región C) utilizando cebadores descritas originalmente por Kojima et al. 5 se detalla. La secuenciación del producto de PCR de la PCR convencional permite la diferenciación de genotipos pertenecientes al GI y GII genogrupos.

Watch this Scientific Journal Video about Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR at JoVE.com

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

Un One-Step RT-PCR para la detección e identificación genogrupo de los aislamientos de Norovirus en las heces de los niños, que utiliza cebadores y sondas TaqMan específicas para el marco de lectura abierto 1 (ORF1)-ORF2 región de la unión, la región más conservada del genoma del norovirus es descrito. Un no-comercial, rentable método de extracción de ARN se detalla.

Detección y Genogrouping de norovirus en las heces de los niños por TaqMan en un solo paso 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

17.1K vistas.  Universidad Peruana Cayetano Heredia. Un One-Step RT-PCR para la detección e identificación genogrupo de los aislamientos de Norovirus en las heces de los niños, que utiliza cebadores y sondas TaqMan específicas para el marco de lectura abierto 1 (ORF1)-ORF2 región de la unión, la región más conservada del genoma del norovirus es descrito. Un no-comercial, rentable método de extracción de ARN se detalla.

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

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.

Detección de microARN en tumores de próstata por el cuantitativo PCR en tiempo real (qPCR)

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

Investigación

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.

Cerenkov Luminiscencia Imaging (CLI) para la Vigilancia del Cáncer Terapia

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.

Implantación de la vena cava inferior interposición del injerto en modelo de ratón

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.

El aislamiento y la inmortalización de líneas celulares derivados de los pacientes a partir de una biopsia muscular para Modelado de Enfermedades

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.

Establecimiento de Human epitelial Enteroids y Colonoids de Whole Tejidos y Biopsia

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.

Infusión del conducto pancreático: un método eficaz y selectivo de administración de fármacos y virus

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.

Aislamiento de hepatocitos primario de ratón para proteína naciente síntesis análisis por el método de etiquetado no radiactivo 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.

Detector de metales portátil detección de ingestión de cuerpo extraño metálico en niños

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.

Detección basada en plaquetas de óxido nítrico en sangre mediante la medición de VASP fosforilación

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.

Evaluación y caracterización de recipientes hialoides en ratones

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