The authors have no acknowledgements.

1. torunda de muestreo en el campo <ol><li> Use un par de guantes limpios. </li><li> Medir el tamaño de la zona de muestreo sin tocar la superficie usando una cinta de medición o una regla. Tratar de estimar el área con la mayor precisión posible y llenar un formulario de informe (Cuadro 1). </li><li> Compruebe el kit de raspado de posibles fugas y bolsas de transporte de muestras de etiquetas y kits de hisopos. </li><li> Mueva el hisopo por el área de muestreo como sigue: un golpe en la dirección horizontal, de un solo golpe en la dirección vertical, y un golpe en una dirección diagonal. No limpie un área superficial mayor que 700 cm 2. </li><li> Coloque cada hisopo en un tubo y apretar bien la tapa. </li></ol> 2. Almacenamiento y Transporte de hisopos al laboratorio <ol><li> Mantenga hisopos a 4 ° C durante un máximo de 48 h. Si se requiere el almacenamiento durante periodos más largos, almacenar los hisopos a -20 ° C (-70 ° C o). </li><li> Mantener los tubos a 0-4 ° C (es decir,. , Utilizar bolsas de hielo) en un recipiente aislado durante el transporte al laboratorio y se envían dentro de las 24-48 horas de la recolección. </li></ol> 3. La concentración de virus, el ARN viral Extracción y Purificación NOTA: Todos los pasos de centrifugación usar una centrífuga de mesa a 5.000 xg durante 5 minutos a temperatura ambiente, a menos que se indique lo contrario. Tenga mucho cuidado cuando se trabaja con el tampón universal de extracción de ácidos nucleicos (UNEX). Use gafas o careta. <ol><li> Etiquetar un tubo de 15 ml y una columna de ARN Midi para cada muestra. Incluir un control negativo de extracción en cada experimento. </li><li> Para preparar la solución de lisis durante 10 hisopos, mezclar 25 ml de tampón de UNEX con 25 ml de PBST (PBS pH 7,2 que contiene 0,02% de Tween-80). </li><li> Añadir 50 l de suspensión de colifago MS2 (10 6 PFU / l) a 50 ml de solución de lisis. </li><li> Coloque una torunda en un tubo de 15 ml y añadir 5 ml de solución de lisis. Mezclar e incubar durante 10 min a temperatura ambiente. </li><li>Añadir 5 ml de etanol 100% a cada tubo y agitar durante 10 s. </li><li> Retire cuidadosamente el hisopo del tubo de 15 ml (que contiene tampón UNEX) presionando suavemente contra el lado del tubo para eliminar el exceso de líquido y luego desechar el hisopo. Los volúmenes restantes deben ser de entre 9-10 ml. </li></ol> 4. Columna Midi Viral Nucleic Acid Extracción <ol><li> Transferir cuidadosamente 4.5 / mezcla de etanol ml UNEX desde el paso 3.6 en una columna de midi, centrifugar y desechar el filtrado. </li><li> Cargar otros 4,5 ml de la misma mezcla en la misma columna, se centrifuga, y desechar el filtrado. </li><li> Para el primer lavado, añadir 3,5 ml de etanol al 70% en la región de Midi columna, centrifugar y desechar el filtrado. </li><li> Para el segundo lavado, añadir otros 3,5 ml de etanol al 70% sobre la columna de Midi. Centrífuga y desechar el filtrado. </li><li> Para el seco-spin, centrifugar las columnas Midi para eliminar todos los restos de etanol (que puede afectar negativamente a su recuperación de ARN viral). </li><li>Colocar la columna de Midi en un nuevo tubo de centrífuga de 15 ml. Añadir 250 l de tampón de elución en la columna de Midi; esperar a 1 min antes de la centrifugación para obtener la mayor recuperación de ARN viral. </li><li> Almacenar el ácido nucleico extraído a -70 ° C o proceder inmediatamente al paso 5. </li></ol> 5. La concentración de ácido nucleico viral utilizando ARN Clean y Concentrador Kits <ol><li> Añadir 500 l de tampón de unión de ARN a 250 l de ARN eluido en el paso 4.7 anterior y agitar durante 10 s. </li><li> Añadir 750 l de etanol 100% y agitar durante 10 s. </li><li> Etiquetar una columna de giro para cada muestra. Cargar la muestra de 750 l en una columna de centrifugado. </li><li> Centrifugar las columnas de centrifugación a 12.000 xg durante 1 min. Descartar el flujo continuo. </li><li> Cargar los 750 l restantes sobre una columna de centrifugación y se centrifuga a 12.000 xg durante 1 min. </li><li> Para el pre-lavado, añadir 400 l de tampón RNA Prep a cada columna de centrifugación y centrifugar a 12.000 xg durante 1 min. Descartar el flujo continuo. </li><li> Para el primer lavado, añadir 800 l de ARN tampón de lavado a cada columna de centrifugación y se centrifuga a 12.000 xg durante 1 min. Descartar el flujo continuo. </li><li> Para el segundo lavado, añadir 400 l de tampón de lavado de ARN para hacer girar la columna y se centrifuga a 12.000 xg durante 1 min. Descartar el flujo continuo. </li><li> Para el seco-spin, centrifugar la columna de centrifugación para eliminar todos los restos de tampón de lavado a 12.000 xg durante 2 min. </li><li> Transferir cuidadosamente la columna de centrifugación a un tubo de microcentrífuga de 1,5 ml limpio. </li><li> Añadir tampón de elución de 25 l en la columna de centrifugación y se incuba durante 1 min a temperatura ambiente. Esto aumentará la recuperación del ARN viral de la columna. </li><li> Recoger ARN por centrifugación la columna de centrifugación a 10.000 xg durante 1 min. </li><li> O bien proceder directamente a RT-qPCR (paso 6) o tienda de ARN a -70 ° C. </li></ol> 6. Mutiplex RT-qPCR Detección de Genogrupo I y II Los norovirus, y colifago MS 2 (Cuadro 2) <ol><li> superficies de trabajo limpias, pipeteeES, y centrífugas con RNasa solución de descontaminación para reducir la posible contaminación. </li><li> Descongelar los reactivos RT-qPCR en hielo. ARN deshielo en hielo (en un área / habitación separada). </li><li> Determinar el número de reacciones y añadir al menos un 10% más de ellos (por ejemplo, si se necesitan 10 reacciones, que mezcla maestra de 11). </li><li> Vortex componentes de la mezcla maestra individuales, a excepción de 25x enzima RT-qPCR, durante 5 s. Mezclar con cuidado la enzima con un movimiento rápido del tubo con un dedo. </li><li> Brevemente componentes de la mezcla centrífuga maestros para 5 s. </li><li> Preparar la mezcla maestra para la detección en tiempo real de RT-PCR de GI norovirus y GII de acuerdo con las instrucciones del kit y añadir cebadores y sondas de oligonucleótidos de norovirus específico en un 1,5 ml tubo de microcentrífuga (Tabla 3). </li><li> Mezclar la mezcla maestra pipeteando 5-10 veces arriba y abajo (no se recomienda agitación). Alícuota de 22 l de la mezcla maestra en cada pocillo de PCR en tiempo real placa de 96 pocillos. </li><li> Vortex ARN de la muestradurante 5 segundos, y cobrar por breve (5 s) centrifugación. </li><li> Añadir 3 l de muestra de RNA y GI y GII controles positivos a la placa de RT-qPCR (siga la plantilla de la Tabla 2). Añadir 3 l de nucleasa freewater en los pozos sin molde y controles (NTC). </li><li> Sellar la placa en tiempo real con película adhesiva óptica. </li><li> centrifugar con cuidado la placa en tiempo real en 1300 xg durante 1 min para eliminar las burbujas de aire o las gotas de líquido que pueden estar presentes en los pocillos. </li><li> Establecer un instrumento en tiempo real PCR y de establecimiento de las siguientes condiciones de termociclado: 1) etapa de RT durante 10 min. a 45 ° C (2) la activación de Taq polimerasa, 10 min. a 95 ° C y (3) 45 ciclos de 15 s a 95 ° C, y 60 s a 60 ° C. </li></ol> 7. La cuantificación de norovirus en muestras de frotis <ol><li> Comprobar los resultados de los controles positivos y negativos para validar los resultados de RT-qPCR (Tabla 3). </li><li> Determina si cada curva estándar se encuentra con elLos valores aceptables dan en la tabla complementaria 2 y calcular la desviación estándar global para la curva estándar utilizando la Ecuación 1. (Ecuación 1)% de eficiencia = 100 x 10 (promedio de valores de Ct-Intercepción) / pendiente. </li><li> Si el software termociclador de PCR no calcula la pendiente de cada curva patrón, determinar la pendiente y los valores R 2 de la regresión utilizando el software estadístico (por ejemplo, Excel, SPSS o SAS). Además, se calcula el% de eficiencia para las curvas de calibración siguiente ecuación I </li><li> Registre el número de copias de ARN calculado por el software termociclador para todas las muestras de prueba en base a las curvas de calibración que cumplen los criterios especificados en la Tabla 4 complementario. Vuelva a ejecutar ninguna de las muestras con las curvas de calibración t sombrero no cumplen con los criterios o tienen controles positivos falsos. Comprobar los valores de Ct de colifago MS2, que se añadió como un control interno para controlar la inhibición de la PCR. </li><li> Determinar el número total de copias de ARN pmuestra er por la multiplicación del número de copias de ARN calculada en la etapa 7.2 por la relación del volumen (25 a 50 l) de eluyente de ARN total a la del ARN (3-5 l) utilizado para la reacción RT-qPCR. Alternativamente, el cálculo de la densidad de RNA dividiendo el número total de copias de ARN por el área de superficie del objeto (cm 2) analizada. </li></ol> 8. El genotipado de muestras en tiempo real RT-PCR positivos por Hemi anidada amplificación por PCR convencional <ol><li> Preparar la primera ronda de mezcla maestra para cada conjunto de cebadores de la RT-PCR ensayo (que complementa los cuadros 2 y 5). </li><li> Añadir 5 l de ARN norovirus positiva a 20 l de mezcla maestra. </li><li> Ejecutar la RT-PCR en las siguientes condiciones: (1) RT paso durante 30 min a 42 ° C (2) la activación de Taq polimerasa, 15 min a 95 ° C, y (3) 40 ciclos de 30 s a 95 ° C , 30 s a 50 ° C y 60 seg a 72 ° C. Después de 40 ciclos, se incuba durante 10 min adicionales a 72 ° C. </li><li>Preparar la segunda ronda de mezcla mater para cada conjunto de cebadores de la RT-PCR ensayo (que complementa los cuadros 2 y 5). </li><li> Añadir 23 l maestro de la mezcla y añadir 2 l de productos de primera ronda de RT-PCR (de primera ronda) a cada tubo (lo ideal es el producto de primera ronda debe ser diluido 1/10 en agua libre de RNasa). </li><li> Repita el paso 8.3. </li><li> Preparar un gel de agarosa al 2% en EDTA 100 ml 1x Tris-acetato (Tris 40 mM, ácido acético 20 mM, y EDTA 1 mM, a pH 8,3). Después de disolver de agarosa por calentamiento de la mezcla en un horno de microondas, añadir 10 l de colorante de ácidos nucleicos por 100 ml de gel preparado después de enfriar la mezcla a 60-70 ° C, se vierte el gel e insertar los peines. Deje que el gel se conforme con al menos 30 minutos. </li><li> Mezclar 15 l de cada producto de RT-PCR con 3 l de colorante de carga 6x. Cargar 15 l de cada muestra y a electroforesis el gel de agarosa a 100 V durante 1 h. </li><li> fragmentos objetivo Impuestos Especiales RT-PCR de tamaño apropiado (330 pb para GI y 341 pb para GII) a partir de un gel de lad purificar ARN usando un kit de extracción de gel comercial. El producto de PCR purificado se puede utilizar ahora para la secuenciación de Sanger. </li></ol>

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Authors have no conflicting interest. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

Los norovirus tienen una dosis infecciosa humana 50% entre 18 y 10 3 20 partículas de virus. Por lo tanto, incluso la contaminación de bajo nivel de las superficies puede suponer un riesgo para la salud pública. Se evaluaron varios aspectos del protocolo de muestreo hisopo incluyendo: 1) diferentes materiales de hisopo, 2) hisopos de condiciones de almacenamiento durante el transporte, 3) la concentración de ARN viral, y 4) colífagos MS2 como el control de la extracción interna. ...

<html> Norovirus humanos son una causa principal de la epidemia y la gastroenteritis aguda esporádica en todo el mundo 1, 2, 3. El virus es muy contagioso y la transmisión ocurre a través de la interacción persona directa a persona o indirectamente por contacto con alimentos contaminados, agua o superficies ambientales. Los norovirus se pueden desprender durante períodos prolongados y prolongan la supervivencia del virus en las superficies ambientales se ha documentado 1, 2, 3. Durante derramamiento pico, miles de millones de partículas de virus son liberadas por gramo de heces, y vómito también contiene un número suficiente de partículas virales para causar la infección 4, 5, 6, 7, 8,ef &quot;&gt; 9, 10. Además, la transferencia del virus entre las superficies inanimadas y la piel humana puede ocurrir fácilmente 2, 11, 12. Por lo tanto, el control de la contaminación ambiental puede ayudar en la investigación de brotes y en la evaluación de la eficacia de la limpieza y procedimientos de desinfección. Varios protocolos de muestreo ambientales se han descrito para la detección de rotavirus, colifago MS2, calicivirus felino (FCV), y bacteriófago P22 13, 14, 15, 16. Sin embargo, las condiciones de validación descritas en estos estudios, incluyendo la desecación rápida (&lt;1 hora) y la escasez de superficie (25 x 100 cm 2), pueden no representar adecuadamente la configuración del campo. Además, el bajos niveles de contaminación de Enviro esperadasuperficies nmental requieren protocolos que son capaces de detectar muy pocas partículas de virus. Hemos desarrollado un método de muestreo de superficie a base de macroespuma para la detección y tipificación de norovirus. Este método ha sido validado durante varios brotes de norovirus. El protocolo incluye 1) cómo recolectar muestras de frotis de superficies ambientales (2) mejor manera de mantener la integridad de las muestras durante la recogida y el envío al laboratorio, y 3) las pruebas de laboratorio y tipificación de norovirus.

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			<td height="21" style="height:21px;width:212px;">Generic name for kits</td>
			<td style="width:259px;"></td>
			<td style="width:153px;"></td>
			<td style="width:185px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Macrofoam swab</td>
			<td>Premoistened EnviroMax Swab kit&#160;</td>
			<td>Puritan</td>
			<td>2588060PFUW</td>
			<td></td>
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			<td height="20" style="height:20px;">&#160;RNA Lysis buffer&#160;</td>
			<td>CDC UNEX buffer</td>
			<td>Microbiologics</td>
			<td>Cat No MR0501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNA extraction spin column</td>
			<td>Midi column</td>
			<td>Omega Biotek</td>
			<td>Cat No R6664-02</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:212px;">RNA purification spin column</td>
			<td>Zymol RNA Clean and Concentrator kit&#160;</td>
			<td>Zymo Research</td>
			<td>Cat No R1016</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Real time RT-PCR kit</td>
			<td>AgPath kit One-Step RT-PCR Kit</td>
			<td>Life Technologies</td>
			<td>Cat No 4387391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conventional RT-PCR kit</td>
			<td>Qiagen one step RT-PCR kit</td>
			<td>Qiagen kit</td>
			<td>Cat No 210212</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Gel extraction kit</td>
			<td>Qiagen QIAquick gel extraction kit</td>
			<td>Qiagen kit</td>
			<td>Cat No 28704 or 28706</td>
			<td></td>
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		<tr height="29">
			<td height="29" style="height:29px;"></td>
			<td>Coliphage MS2</td>
			<td>ATCC</td>
			<td>Cat No 15597-B1</td>
			<td></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;"></td>
			<td>RNA run-off transcripts</td>
			<td></td>
			<td></td>
			<td style="width:285px;">Bacteriophage MS2 (ATCC No. 15597-B1) can be cultivated using Escherichia coli (E.coli) Famp (ATCC No. 700891).&#160;</td>
		</tr>
		<tr height="148">
			<td height="148" style="height:148px;"></td>
			<td>Realtime PCR platform</td>
			<td>Applied Biosystems</td>
			<td>Model ABI 7500</td>
			<td style="width:285px;">GI and GII RNA run off transcripts were quantified spectrophotometrically at A260, diluted in diethyl pyrocarbonate-treated water to 1 &#215; 106 copies/ &#956;l, and stored at &#8722;80&#176;C with 1.0 U /&#956;l RNasin (Promega, Madison, WI).&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td>Optical 96-well reaction plate</td>
			<td>Thermo Scientific</td>
			<td>Cat No 4316813</td>
			<td></td>
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			<td height="22" style="height:22px;"></td>
			<td><a href="http://www.thermofisher.com/order/catalog/en/US/adirect/%20https:/www.thermofisher.com/order/catalog/en/US/adirect/lt?cmd=catProductDetail&amp;showAddButton=true&amp;productID=4306311&amp;_bcs_=H4sIAAAAAAAAAI1RTWvDMAz9Nb4srDg2yXpNW3rZVyHbuRhHSQx2HWwnJf9%2BUkO6scMYGEmWnqX3%0A5Mec8e0p%2BGbUKWZMlFkNYTIa4h%2F5PqWByYqJI57r9bpJPQTnWxPRb7R3mB4jGrig6b0DdD40WOyT%0As9iBCUmHb1MYge78iYi06ZznPEd0exacF6LkD5h%2BNTr4yg1II0DH5C6rmh6imSA7GuuInsIq5TUy%0AjD6Yf%2FKU1Zav3NBrlZT13cr8s0ajGhNAJ4xsYvKoXcPkAYEHEwer5jrNFuWVdF1ieajNpbPwAhPY%0AvUrQ%2BTAjAt88w4zlm8Ly%2FYRha2yiyeUSUJM35ajHt%2BZiUS0KuWNityrHkLSjuyHUDbkgfuxgbfwx%0AD9Q0vydo9rrs4te675%2FTKhvpd74AhB0C9yUCAAA%3D&amp;returnURL=http%3A%2F%2Fwww.thermofisher.com%3A80%2Forder%2Fcatalog%2Fen%2FUS%2Fadirect%2Flt%3Fcmd%3DcatDisplayStyle%26catKey%3D101%26filterType%3D1%26OP%3Dfilter%26filter%3Dft_1101%252Ff_2005260*">MicroAmp Clear Adhesive Film&#160;</a></td>
			<td>Thermo Scientific</td>
			<td>Cat No 4306311</td>
			<td></td>
		</tr>
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swab sampling method for the detection of human norovirus on surfaces

norovirus humanos son una causa principal de la epidemia y la gastroenteritis esporádica en todo el mundo. Debido a que la mayoría de las infecciones se propagan ya sea directamente a través de la ruta de persona a persona o indirectamente a través de las superficies ambientales o alimentos, fómites contaminados y superficies inanimadas son vehículos importantes para la propagación del virus durante los brotes de norovirus. Desarrollamos y evaluamos un protocolo utilizando hisopos macroespuma para la detección y tipificación de los norovirus humanos de las superficies duras. En comparación con hisopos con punta de fibra o toallitas antiestáticas, hisopos macroespuma permiten la recuperación de virus (rango de 1,2 a 33,6%) de las superficies de asiento de inodoro de hasta 700 cm 2. El protocolo incluye los pasos para la extracción del virus de los hisopos y una mayor concentración del ARN viral usando columnas de centrifugación. En total, 127 (58,5%) de 217 muestras de frotis que habían sido recogidos de las superficies en los cruceros y centros de atención a largo plazo donde había estado la gastroenteritis por norovirusinformaron dado positivo por norovirus GII por RT-qPCR. De estos 29 (22,8%) podría ser genotipo con éxito. En conclusión, la detección de norovirus en superficies ambientales utilizando el protocolo que hemos desarrollado puede ayudar a determinar el nivel de contaminación del medio ambiente durante los brotes, así como la detección de virus cuando las muestras clínicas no están disponibles; también puede facilitar el seguimiento de la eficacia de las estrategias de remediación.

La Figura 1 presenta un diagrama de flujo del protocolo de muestreo hisopo. Este protocolo consiste en cuatro pasos principales; 1) la recogida de muestras, 2) de almacenamiento de muestras y el transporte, 3) la purificación de ARN viral y la concentración y 4) ensayo de RT-qPCR y genotipado. <img alt="Figura 1" src="/files/ftp_upload/55205/55205fig1.jpg" /> Figura 1: Diagrama de flujo del prot...

Watch this Scientific Journal Video about Swab Sampling Method for the Detection of Human Norovirus on Surfaces at JoVE.com

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Método de muestreo hisopo para la Detección de norovirus humano en superficies

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

swab-sampling-method-for-the-detection-of-human-norovirus-on-surfaces

Research

JoVE Journal

Immunology and Infection

19.5K vistas.  Centers for Disease Control and Prevention. A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces. 

Video: Swab Sampling Method for the Detection of Human Norovirus on Surfaces

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

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

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

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

Combinación de adhesivo basado en cinta de muestreo y de fluorescencia In situ La hibridación para la detección rápida de Salmonella En los productos frescos

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

Investigación

Inmunología e Infección

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

Punción en el dedo de sangre Metodología de muestreo para la determinación de la inducida por el ejercicio apoptosis de los linfocitos

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

ampliPHOX colorimétrico de detección de un microarray de ADN para la influenza

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

High-throughput Detection Method for Influenza Virus

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

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

De alto rendimiento Método de detección de virus de la influenza

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

Placa de Ensayo para Norovirus murino

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

Super-resolución de imagen de la célula Natural Killer inmunológica Synapse en un apoyada-Glass planar bicapa lipídica

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Los ensayos cualitativos y cuantitativos para la detección y caracterización de proteínas de antimicrobianos

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

Método del agitador magnético para la detección de<em&gt; Trichinella</em&gt; Las larvas en muestras de músculo

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

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

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

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

La neuraminidasa Ensayo de inhibición basado en la fluorescencia para evaluar la susceptibilidad de los virus de la influenza a El Inhibidor de la neuraminidasa clase de antivirales

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

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

Muestreo no invasivo de revestimiento mucoso líquido para la cuantificación In Vivo de la vía aérea superior mediador inmune niveles

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...