Esta labor fue apoyada por los Institutos Nacionales de Salud (NIH) en AI089106 RC3 subvención. Ácidos nucleicos MDR-TB fueron proporcionados por / Programa Especial de las Naciones Unidas para el Fondo / Naciones Unidas para el Desarrollo del Banco Mundial / Organización Mundial de la Salud para la Investigación y Capacitación en Enfermedades Tropicales (TDR), Ginebra, Suiza. Agradecemos al Dr. Tom Shinnick de los Centros para el Control y la Prevención de Enfermedades de orientación sobre los genes y las mutaciones específicas para incluir en el ensayo de prototipos.

Para los laboratorios que siguen las precauciones universales de PCR, es operacionalmente más eficiente para incluir varios microarrays de amplificación y juntas por sustrato y lavar todos los microarrays de amplificación simultánea en un recipiente a granel, tal como se describe aquí. Formatos consumibles están disponibles para la realización de post-amplificación pasos de microarrays se lavan en una, prueba amplicón cerrado completamente sellada, según ha informado en otras partes 30,33. 1. Configuración <ol><li> Extraer y purificar ácidos nucleicos a partir de la muestra bajo condiciones de bioseguridad apropiadas con un método de elección. El requisito es que los ácidos nucleicos sean de pureza suficiente para la amplificación asimétrica, PCR multiplex. </li><li> Preparar espacio de trabajo limpiando las superficies y equipos con las soluciones descontaminantes. </li><li> Coloca los reactivos de amplificación en hielo o bloque frío. </li><li> Etiquete un tubo de microcentrífuga de 1,5 ml estéril para la mezcla maestra de amplificación, y la etiqueta de un estéril de 0,2 ml de microcentrífuga maser para cada muestra. </li><li> Después de reactivos se descongelan, vórtice brevemente y recogen los contenidos de la parte inferior del tubo con un pulso de espín en un mini-centrífuga. No agite Taq polimerasa. Golpee suavemente el tubo para mezclar y siga con un pulso-spin en mini-centrífuga. </li><li> Preparar una mezcla maestra de amplificación utilizando los volúmenes de reacción por-ejemplo que se muestra en la Tabla 1. Para tener en cuenta posibles inexactitudes de pipeteado, preparar al menos un volumen de reacción más que el número total de muestras que se están procesando. Tenga en cuenta que la mezcla maestra contiene Taq polimerasa adicional, más allá de lo que se proporciona con el tampón de PCR muliplex. Sólo agregar el control de amplificación / inhibición de la mezcla maestra después se combinan todos los demás reactivos, y los tubos de reactivo stock devueltos al almacenamiento. </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Reactivo </td><td> Volumen por muestra (l) </td><td> &lt;strong&gt; final [] </td></tr><tr><td> Multicine PCR Buffer con HotStar Taq Plus </td><td> 25 </td><td> 1x </td></tr><tr><td> Albúmina de suero bovino (BSA) </td><td> 0.55 </td><td> 0,6 mg / ml </td></tr><tr><td> Formamida </td><td> 3.8 </td><td> 7,6% </td></tr><tr><td> Taq polimerasa adicional </td><td> 0.8 </td><td> unidades / l (4 unidades en total) </td></tr><tr><td> Mezcla de cebadores MDR-TB </td><td> 15.75 </td><td> - </td></tr><tr><td> RNasa libre de H 2 O </td><td> 2.1 </td><td> - </td></tr><tr><td> La amplificación / control de inhibición </td><td> 1 </td><td> 5,0 fg / l </td></tr><tr><td> Total </td><td> 49 </td><td></td></tr></tbody></table> Tabla 1. MDR-TB de microarrays de amplificación composición mezcla maestra. <ol start="7"><li> Vortex la mezcla maestra y recoger contenidos a la parte inferior del tubo con un pulso de espín en un mini-centrífuga. </li><li> Combine 49 l de mezcla maestra y 1 l de M. tuberculosis ADN a cada uno de los tubos de muestra de la etapa 1.4, más arriba. Para una externa, sin control de la plantilla (NTC), use 1 l de agua grado biología molecular en lugar de la M. muestra de ADN de la tuberculosis. Vortex y el pulso y gire el tubo (s) cuando haya terminado. </li></ol> 2. Carga de amplificación Microarrays <ol><li> Añadir 48 l de cada master mix / muestra en el centro de sus respectivos microarrays, teniendo cuidado de no tocar el mismo microarray. </li><li> Coloque una hoja de la cubierta en la parte superior de la junta de microarrays y el sello, con cuidado de no atrapar burbujas de aire en la cámara de microarrays. Las burbujas de aire pueden afectar negativamente a la eficiencia de amplificacióny pueden crear artefactos o ruido en la imagen de microarrays posterior. </li></ol> 3. Ciclo térmico <ol><li> Una vez que todos los microarrays se cargan con la muestra, lugar sustratos en el termociclador bloque plano. Asegúrese de que la opción de tapa térmica se encuentre en &quot;Off&quot;. Equipo obtenida de Akonni Biosystems ya estará precalificada para su uso. De lo contrario, es muy importante verificar que el termociclador bloque plano (s) proporcionan (s) uniforme, calefacción consistente a través de todos los sustratos de microarrays. </li><li> Abra el software termociclador, seleccione el programa apropiado, escriba &quot;50 l&quot; para el volumen de reacción, e iniciar la carrera. El protocolo de ciclado térmico descrito aquí consiste en: <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Pasos del ciclo termal </td></tr><tr><td> 1 </td><td> 88 ° C </td><td> 5 min </td></tr><tr><td> 2 </td><td> 88 ° C </td><td> 30 seg </td></tr><tr><td> 3 </td><td> 55 ° C </td><td> 1 min </td></tr><tr><td> 4 </td><td> 65 ° C </td><td> 30 seg </td></tr><tr><td> 5 </td><td> Repita los pasos (2-4) durante 50 ciclos </td></tr><tr><td> 6 </td><td> 65 ° C </td><td> 3 min </td></tr><tr><td> 7 </td><td> 55 ° C </td><td> 3 hr </td></tr></tbody></table></li><li> El número total de ciclos térmicos y de 55 ° C después de la amplificación de tiempo de espera son variables independientes que el usuario puede modificar, según sea necesario o apropiado para el experimento específico. Debido a que la mezcla maestra de la MDR-TB crea amplicones asimétricos (predominantemente de cadena sencilla), por lo general es útil para incluir más ciclos térmicos que de otro modo podrían ser utilizados para un protocolo convencional, exponencial de PCR de amplificación. </li><li> Cuando el programa de ciclos y la hibridación térmica es completa, terminar el programa, retire los microarrays de amplificación, cierre el software y apague el termociclador (s).</li></ol> 4. Lavar y seco <ol><li> Para el lavado de hasta 24 sustratos simultáneamente, preparar al menos 250 ml de SSPE 1x - 0,1% de Triton X-100 de tampón de lavado, y dos contenedores con al menos 250 ml de agua desionizada o agua Milli-Q grado cada uno. El tampón de lavado puede ser preparado de antemano. </li><li> Retire con cuidado la hoja de la cubierta y la junta de cada cámara de amplificación de microarrays utilizando pinzas de gama plana. Este paso es el punto en el que existe el mayor riesgo de dañar físicamente a conjuntos de elementos de gel. Utilice los controles adecuados de contaminación de PCR para evitar la propagación de los ácidos nucleicos amplificados dentro del entorno de trabajo. </li><li> Coloque sustratos de microarrays en un soporte de diapositivas histología, y coloque todas las diapositivas en el cubo de lavado que contiene el tampón de lavado. Cubrir el recipiente con una tapa. </li><li> Lavar microarrays durante 10 min a temperatura ambiente con agitación suave. </li><li> Sumerja los microarrays tres veces en cada una de dos lavados sucesivos del desionizada oAgua de calidad Milli-Q, y secar al aire. </li></ol> 5. Imaging La mezcla de cebadores MDR-TB asimétrica genera amplicones etiquetados con Cy3. Microarrays elementos Gel se pueden obtener imágenes con cualquier reproductor de imágenes de microarrays estándar capaz de Cy3 imágenes. El siguiente procedimiento de imagen es específico para la mezcla de la MDR-TB maestro, campo Dx2100 generador de imágenes portátil, y el software de análisis automatizado proporcionado por Akonni. <ol><li> Asegúrese de que el cable Ethernet de la cámara se conecta al ordenador. </li><li> Encienda el reproductor de imágenes. El interruptor de encendido se encuentra en el panel posterior del instrumento. LEDs azules en la parte frontal de la cámara termográfica se encienden cuando cámara se enciende. </li><li> Encienda el ordenador. </li><li> En el escritorio del ordenador, haga doble clic en el icono del software para iniciar el software automatizado de análisis de imágenes. </li><li> Esperar a que el software para establecer una conexión con la cámara de imágenes. Una vez se establece la conexión, el botón Analizar esté disponible, y elmensaje &quot;Conexión correcta&quot; aparecerá en la esquina inferior izquierda de la pantalla. </li><li> Insertar el microarray de amplificación se secó con el microarray frente lejos del usuario, y hacia la lente del objetivo. Si el microarray está orientada de forma incorrecta con respecto a la lente y la cámara, el software de análisis automatizado fallará para colocar una cuadrícula en la imagen de fluorescencia. </li><li> Cierre la puerta de acceso. Una imagen de vista previa estará disponible en la pantalla de ordenador. </li><li> Seleccione la MDR-TB script de análisis desde el menú desplegable e introduzca el tiempo de exposición deseado (en milisegundos). Un punto de partida típico para elementos gel microarrays de amplificación es 100-500 ms. </li><li> Píxeles saturados se mostrarán en la Vista preliminar, Imagen en color rojo. Ajustar el tiempo de exposición para reducir al mínimo el número de píxeles saturados dentro de manchas individuales. </li><li> Haga clic en Analizar para adquirir y analizar la imagen de fluorescencia. El software automáticamente colocará unMDR-TB grid-ensayo específico sobre la imagen, extraer intensidad integrada y valores de fondo, e informar los resultados de resistencia a los medicamentos y de genotipificación. El análisis automatizado normalmente tarda unos segundos. Para imágenes desafiantes que contienen rayaduras, polvo, artefactos fluorescentes, o daño físico a las características de microarrays, el software puede necesitar hasta 1 min para completar el análisis. </li><li> Una vez terminado el análisis, haga clic en Guardar, seleccione la carpeta de destino, escriba un nombre de archivo y haga clic en Aceptar. Microarrays de datos subyacentes se guardan como un archivo xls. Y pueden ser exportados para análisis fuera de línea, si lo desea. </li><li> Una vez que el análisis haya finalizado, retire los microarrays de amplificación de la cámara termográfica, y haga clic en Nuevo para iniciar el siguiente análisis. </li><li> Cuando haya terminado la recolección de datos, cierre el software, apague el ordenador y apague el reproductor de imágenes. </li></ol>

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Microbiol. 45, 2802-2807 (2007).</li><li>Chandler, D. P., Bryant, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+amplification+microarrays+for+infectious+disease+diagnostics.">Integrated amplification microarrays for infectious disease diagnostics.</a> Microarrays. 1, 107-124 (2012).</li><li>Vincent, V., Rigouts, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TDR+Tuberculosis+Strain+Bank:+a+resource+for+basic+science,+tool+development+and+diagnostic+services.">The TDR Tuberculosis Strain Bank: a resource for basic science, tool development and diagnostic services.</a> Int. J. Tuberc. Lung Dis. 16, 24-31 (2012).</li><li>Golova, J. B., Chernov, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-volatile+copolymer+compositions+for+fabricating+gel+element+microarrays.">Non-volatile copolymer compositions for fabricating gel element microarrays.</a> Anal. Biochem. 421, 526-533 (2012).</li><li>Cooney, C. G., Sipes, D., Thakore, N., Holmberg, R., Belgrader, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+plastic,+disposable+microfluidic+flow+cell+for+coupled+on-chip+PCR+and+microarray+detection+of+infectious+agents.">A plastic, disposable microfluidic flow cell for coupled on-chip PCR and microarray detection of infectious agents.</a> Biomed. Microdevices. 14, 45-53 (2012).</li><li>Lane, S., Evermann, J., Loge, F., Call, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplicon+secondary+structure+prevents+target+hybridization+to+oligonucleotide+microarrays.">Amplicon secondary structure prevents target hybridization to oligonucleotide microarrays.</a> Biosens. Bioelectron. 20, 728-735 (2004).</li><li>El-Hajj, H. H., Marras, S. A. E., et al. <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+sloppy+molecular+beacon+probes+for+identification+of+Mycobacterial+species.">Use of sloppy molecular beacon probes for identification of Mycobacterial species.</a> J. Clin. Microbiol. 47, 1190-1198 (2009).</li><li>Boehme, C. C., Nabeta, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+molecular+detection+of+tuberculosis+and+rifampin+resistance.">Rapid molecular detection of tuberculosis and rifampin resistance.</a> N. Engl. J. Med. 363, 1005-1015 (2010).</li></ol>

Los autores declaran no tener intereses financieros en competencia.

La medida de multiplexación con una micromatriz de amplificación es dictado en última instancia por la eficiencia de la PCR asimétrica múltiplex, no el microarray. En nuestra experiencia, 10 a 12 pares de cebadores únicos pueden ser multiplexados fácilmente en un formato de microarrays de amplificación. Por lo tanto, criterios de cebador y sonda de diseño convencionales se aplican a los nuevos ensayos, excepto que también se debe tener en cuenta las posibles interacciones entre ácidos nucleicos en fase de sol...

La detección temprana de los casos y el tratamiento rápido se consideran las estrategias de control más eficaces de reducir el Mycobacterium tuberculosis (MTB) de transmisión de 1, y existe un amplio consenso en la comunidad TB que un punto de atención (PDA) o cerca de la prueba POC para diagnosticar simultáneamente TB y se necesita resistencia a los medicamentos (DR). Tecnologías tales como GeneXpert de Cepheid y otras pruebas de amplificación de ácidos nucleicos reducen el tiempo de diagnóstico para muchos pacientes de TB, y proporcionan una rápida lectura de salida que indica la resistencia a la rifampicina o mutaciones seleccionadas que confieren resistencia a otros fármacos de línea primero o segundo 2. Aunque las pruebas de amplificación en tiempo real y de ácidos nucleicos isotérmica están diseñadas para identificar las mutaciones de resistencia a las drogas que llevan a la MDR-TB, el espectro de mutaciones se detectan es a menudo insuficiente para diseñar un régimen individualizado de drogas correspondiente al perfil de resistencia a los medicamentos del paciente, y las limitaciones técnicas relacionadasa la diafonía óptica o la complejidad de la amplificación y de información química 3 a 7 may limitar el número de loci o mutaciones que se detectan. Por lo tanto, se requieren tecnologías de detección con mayor capacidad de multiplexación para subsanar las deficiencias conocidas en el diagnóstico POC MDR-TB. Microarrays y los ensayos con sondas de línea Hain avaladas por la OMS pueden hacer frente a la &quot;múltiples genes, mutaciones múltiples&quot; reto de diagnosticar la TB-MR 8-29. Por desgracia, estas plataformas de detección de multiplexado basado en la hibridación utilizan múltiples pasos, complicado y protocolos de amplificación de voces que exigen la formación y la competencia significativa en las técnicas moleculares. El microarray de amplificación 30 fue diseñado para hacer frente a algunos de estos microarrays de flujo de trabajo y las preocupaciones operacionales. Los principios de fluidos simplificadoras son para amplificar, hibridar, y detectar dianas de ácido nucleico dentro de una cámara única de microfluidos. El usuario introduce los mas ácidos nucleicos y amplificaciónter la mezcla en una cámara de fluido con una pipeta y se inicia el protocolo de ciclado térmico. Para el método de procesamiento por lotes que se muestra aquí, los microarrays se lavan posteriormente en solución en masa, se secaron, y la imagen. Este estudio demuestra la funcionalidad de un microarray de amplificación utilizando una prueba de MDR-TB de microarrays para rpoB (30 mutaciones), katG (2 mutaciones), inh (4 mutaciones), rpsL (2 mutaciones), embB (1 mutación), IS1245, IS6110 , y una amplificación interna y el control de hibridación. Al menos un par emparejado de microarrays sondas (de tipo salvaje (WT) y mutantes de un solo nucleótido (MU)) se incluye para cada mutación de interés. Ácidos nucleicos purificados a partir de M. resistente a múltiples fármacos tuberculosis son de la cepa TDR Tuberculosis Banco 31. Microarrays de elemento en gel se fabrican sobre substratos de vidrio por copolimerización esencialmente como se describe en otra parte 32, excepto que se utiliza 4% de monómero y 0,05 mM de cada sonda en el polimerizablesmezcla ación. Las matrices están rodeados con una junta de 50 ml antes de su uso. Después de ciclos térmicos, de hibridación y de lavado de pasos, microarrays de amplificación se crean imágenes en un analizador portátil Akonni. , La intensidad de señal integrados antecedentes corregido se obtienen de la prima. Imágenes tif utilizando un algoritmo círculo fijo. De ruido para cada elemento de gel se calcula como tres veces la desviación estándar de los fondos contado locales. Objetivos de genes típicamente se consideran detectables durante la relación señal-ruido (SNR) valores ≥ 3. Con el fin de determinar la presencia o ausencia de una mutación específica en cada gen o codón, una relación discriminante se calcula a partir de los valores de SNR como (WT-MU) / (wt + MU). Ratios de discriminantes &lt;0 son indicativos de una mutación de resistencia a fármacos en el locus, mientras que las relaciones&gt; 0 son indicativos de la secuencia de tipo salvaje.

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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;">MDR-TB amplification microarrays, with applied gasket and pre-cut cover slips</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
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			<td height="22" style="height:22px;">Multiplex PCR kit, containing 2X PCR buffer with HotStar Taq plus</td>
			<td>Qiagen</td>
			<td>#206143</td>
			<td></td>
		</tr>
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			<td height="22" style="height:22px;">Taq polymerase</td>
			<td>Qiagen</td>
			<td>#201207</td>
			<td></td>
		</tr>
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			<td height="22" style="height:22px;">RNAse-free water</td>
			<td>Qiagen</td>
			<td>#206143&#160;</td>
			<td></td>
		</tr>
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			<td height="22" style="height:22px;">Formamide</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP227-500</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">20 mg mL-1 non-acetlyated bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>#3B6917</td>
			<td></td>
		</tr>
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			<td height="22" style="height:22px;">5X concentrated MDR-TB primer mix</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">500 fg uL-1 amplification and inhibition control</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">20X SSPE</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP1328-4</td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:22px;">Triton X-100</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP151-500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Disinfecting Spray&#160;</td>
			<td>Current Technologies, Inc.</td>
			<td>#BRSPRAY128</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">70% Isopropyl Alcohol</td>
			<td>Decon Labs, Inc.</td>
			<td>#8416</td>
			<td></td>
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			<td height="22" style="height:22px;">Equipment</td>
			<td>Company&#160;</td>
			<td>Catalog Number&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Microarray imager, with automated image and data analysis software</td>
			<td>Akonni Biosystems</td>
			<td>100-20011</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Thermal cycler with flat block insert</td>
			<td>Akonni Biosystems</td>
			<td>100-10021</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">High-throughput wash station and slide holder</td>
			<td>ArrayIt</td>
			<td>HTW</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Dissecting forceps</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#10-300</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Mini Vortexer</td>
			<td>VWR</td>
			<td>#3365040</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Mini-centrifuge</td>
			<td>VWR</td>
			<td>#93000-196</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Airbrush Compressor Kit</td>
			<td>Central Pneumatic</td>
			<td>#95630</td>
			<td></td>
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demonstrating a multi-drug resistant mycobacterium tuberculosis amplification microarray

La simplificación de los microarrays de flujo de trabajo es un primer paso necesario para la creación de la MDR-TB diagnósticos basados ​​en microarrays que se pueden utilizar de forma rutinaria en los entornos de bajo recursos. Un microarray amplificación combina la amplificación asimétrica PCR, la selección de tamaño de destino, objetivo de etiquetado y la hibridación de microarrays en una única solución y en una sola cámara de microfluidos. Un método de procesamiento por lotes se demuestra con una mezcla maestra asimétrica 9 compleja y de baja densidad elemento gel microarray para el genotipado de múltiples fármacos de Mycobacterium tuberculosis (MDR-TB). El protocolo descrito aquí se puede completar en 6 horas y proporcionar genotipado correcto con al menos 1000 equivalentes celulares de ADN genómico. La incorporación en el chip pasos de lavado es viable, lo que resultará en un método de amplificación totalmente cerrado y el sistema. La medida de multiplexación con una micromatriz de amplificación se ve limitada en última instancia por el número de pares de cebadores que se pueden combinar en un único maestro mIX y aún así lograr la sensibilidad deseada y métricas de rendimiento especificidad, en lugar del número de sondas que están inmovilizados en la matriz. Del mismo modo, el tiempo total de análisis se puede acortar o alargar dependiendo del uso específico previsto, pregunta de investigación, y los límites de detección deseado. Sin embargo, el enfoque general simplifica significativamente microarrays de flujo de trabajo para el usuario final, reduciendo el número de pasos manualmente intensivos y que consumen mucho tiempo de procesamiento, y proporciona un camino bioquímico y de microfluidos simplificado para la traducción de diagnósticos basados ​​en microarrays en la práctica clínica de rutina.

Análisis de imágenes cualitativa puede dar una idea de las fuentes de ruido experimental o variabilidad que son difíciles de identificar en las tablas de datos generados por el software automatizado de análisis de imágenes. Por lo tanto, puede ser útil para comprobar visualmente que 1) todos los elementos de gel están intactos y en buen estado, 2) el fondo mundial está libre de artefactos fluorescentes que pueden afectar señal individual a ruido (SNR) valores, 3) no hay evidencia para la formación de burbujas ...

Watch this Scientific Journal Video about Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray at JoVE.com

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Un microarray de la amplificación de PCR asimétrica combina la amplificación y la hibridación de microarrays en una sola cámara, lo que simplifica significativamente microarrays de flujo de trabajo para el usuario final. La simplificación de los microarrays de flujo de trabajo es un primer paso necesario para la creación de diagnósticos basados ​​en microarrays que se pueden utilizar de forma rutinaria en los entornos más bajos recursos.

Demostración de un resistente a múltiples fármacos<em&gt; Mycobacterium tuberculosis</em&gt; Microarray Amplificación

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

demonstrating-multi-drug-resistant-mycobacterium-tuberculosis

Research

JoVE Journal

Immunology and Infection

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

12.5K vistas.  Akonni Biosystems, Inc..  Un microarray de la amplificación de PCR asimétrica combina la amplificación y la hibridación de microarrays en una sola cámara, lo que simplifica significativamente microarrays de flujo de trabajo para el usuario final. La simplificación de los microarrays de flujo de trabajo es un primer paso necesario para la creación de diagnósticos basados ​​en microarrays que se pueden utilizar de forma rutinaria en los entornos más bajos recursos. 

Video: Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

Pruebas de susceptibilidad antimicrobiana de Mycobacterium tuberculosis complex para drogas de primera línea y la segunda por dilución en caldo en un formato de placa de microtitulación

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

Investigación

Inmunología e Infección

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Preparación de la muestra de<em&gt; Mycobacterium tuberculosis</em&gt; Extractos de Estudios de Resonancia Magnética Nuclear metabolómica

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Crecimiento de Mycobacterium tuberculosis Las biopelículas

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

Mal plegamiento de proteínas priones de amplificación cíclica

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Un modelo experimental para estudiar la tuberculosis-Malaria Coinfección sobre Transmisión Natural de Mycobacterium tuberculosis Y Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

Un nuevo enfoque para la recuperación Microdissection<em&gt; Mycobacterium tuberculosis</em&gt; Las transcripciones específicas de formalina parafina fijo Embedded pulmón granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Un modelo de tejido 3D Pulmón humano de Estudios funcionales en<em&gt; Mycobacterium tuberculosis</em&gt; Infección

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Sistema para la eficacia y la citotoxicidad de detección de inhibidores de focalización intracelular<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

Un ensayo compatible de alto rendimiento para evaluar la eficacia del fármaco contra los macrófagos a pases<em&gt; Mycobacterium tuberculosis</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Análisis de 18FDG PET/CT la proyección de imagen como una herramienta para estudiar la tuberculosis del Mycobacterium infección y tratamiento en Primates no humanos

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...