Esta investigación fue apoyada por el Centro de Investigación Médica de la Marina, la unidad de trabajo de investigación 6000.RAD1.J.A0310. Las opiniones expresadas en este artículo son las de los autores y no reflejan necesariamente la política oficial o la posición del Departamento de la Marina, el Departamento de Defensa, o el gobierno de Estados Unidos. Wei Mei-Ching es un empleado del gobierno de Estados Unidos. Este trabajo fue preparado como parte de sus funciones oficiales. Título 17 USC §105 establece que &quot;La protección de copyright bajo este título no está disponible para toda la obra del Gobierno de los Estados Unidos. &#39; Título 17 USC § 101 define una obra gobierno de Estados Unidos como un trabajo preparado por miembro del servicio militar o empleado del gobierno de Estados Unidos como parte de las funciones oficiales de esa persona.

1. Preparar diluciones de ADN plásmido como estándar para la reacción LAMP <ol><li> Use un espectrofotómetro para medir la densidad óptica (DO) a 260 nm para determinar el plásmido (que contiene IS1111a gen diana de C. burnetii RSA 493) la concentración de ADN. Un OD 260 de 1 equivale a 50 g / l de ADN. </li><li> Convertir la cantidad de ADN plásmido en el número de copias. Dado que el tamaño del plásmido es 6330 pb, el peso molecular de este plásmido es 4,1 x 10 6 g (6.330 pb x 649 g / bp). La concentración de ADN del plásmido es 34,2 ng / l (como se determina a partir del paso 1.1). 1 l de esta muestra de ADN contiene 34,2 ng de ADN plásmido, lo que equivale a 5 x 10 9 copias (34,2 x 10 -9 g / 4,1 x 10 6 gx 6,02 x 10 23) de ADN. </li><li> Añadir 10 l de ADN plásmido (5 x 10 9 copias / l) a 90 l de agua para una dilución de 10 veces para hacer una muestra de ADN de 5 x 10 8 copias / l. </li><li> Repita el paso 1.3 para preparar muestras de ADN de 5 x 10 7, 5 x 10 6, 5 x 10 5, 5 x 10 4, 5 x 10 3, 5 x 10 2, 5 x 10 1, y 5 x 10 0 copias / l. </li></ol> 2. Prepare la plantilla de ADN de muestras para la reacción LAMP <ol><li> Extraer ADN de muestras de plasma humano usando un kit Mini comercial de ADN de acuerdo con el protocolo del fabricante. Use un total de 200 muestras l para la extracción y eluir el ADN extraído en un volumen de 20 l. </li></ol> 3. Preparar 2x tampón de reacción LAMP se <ol><li> Mezclar 200 l de 10x tampón de Pol, 320 l de 5 M trimetilglicina, 12 l de 1 M MgSO 4, 280 l de mezcla de dNTP (10 mM cada uno) y 188 l de agua para hacer 1 ml de tampón 2x LAMP. Mezcle en un vórtex durante 10 s. </li></ol> 4. Realizar la lámpara estándar de Reacción <ol><li> Mezclar 12,5 l de 2x LATampón MP (tal como se preparó en el paso 3; 40 mM Tris-HCl (pH 8,8), KCl 20 mM, 16 mM MgSO4, 20 mM (NH 4) 2 SO 4, 0,2% de Triton X-100, trimetilglicina 1,6 M, 1,4 mM mezcla dNTP), 1,2 l de mezcla de cebador, 1 l de ADN polimerasa Bst (8 U / l), y 5,3 l de agua para hacer la mezcla de reacción (20 l) en un tubo de PCR de 0,2 ml. </li><li> Añadir 5 l de ADN (del paso 1 o 2) a la mezcla de reacción de 20 l y mezclar bien. </li><li> Cerrar el tubo e incubar a 60 ° C durante 60 minutos en un baño de agua o bloque de calentamiento. Esta es la temperatura óptima de reacción previamente determinada. </li><li> Añadir 5 l de 10x tampón de carga de gel para terminar la reacción. </li><li> Cargar 5 l de productos de reacción en un gel de agarosa al 2% teñido con intercalantes colorante de ácidos nucleicos. </li><li> gel a 100 V correr durante 35 minutos en tampón 1x TBE. </li><li> Visualizar los resultados por la luz UV. </li></ol> 5. Realizar reacción LAMP con reconstituidoreactivos <ol><li> Mezclar 125 l de 10x tampón, Pol 200 l de 5 M trimetilglicina, 7,5 l de 1 M MgSO 4, 667,5 l de agua para hacer 1 ml de tampón de reconstitución. Mezcle en un vórtex durante 10 s. </li><li> Retire los tubos que contienen los reactivos liofilizados de la bolsa de papel de aluminio sellado. No utilizar los tubos si la bolsa de papel de aluminio no está cerrado o si está dañado. </li><li> Añadir 20 l de tampón de reconstitución en cada tubo para resuspender los reactivos liofilizados. </li><li> Mezclar pipeteando arriba y abajo de tampón 5 veces. Echar un vistazo para asegurarse de que los reactivos liofilizados están completamente re-suspendido. El polvo blanco debe desaparecer en el tubo. </li><li> Añadir 5 l de molde de ADN (de la etapa 1 o 2) a los reactivos reconstituidos y mezclar bien. </li><li> Cerrar el tubo e incubar a 60 ° C durante 60 minutos en un baño de agua o bloque de calentamiento. </li><li> Añadir 5 l de 10x tampón de carga de gel para terminar la reacción. </li><li> De carga 5 l de reacciónproductos a un gel de agarosa al 2% teñido con intercalantes colorante de ácidos nucleicos. </li><li> gel a 100 V correr durante 35 minutos en tampón 1x TBE. </li><li> Visualizar los resultados por la luz UV. </li></ol> 6. Realizar LAMP reacción con tampón de reconstitución que contiene HNB fluorescente o colorante intercalante <ol><li> Mezclar 125 l de 10x de Pol Buffer, 200 l de 5 M trimetilglicina, 7,5 l de 1 M MgSO 4, 7,5 l de HNB 20 mM (o 12,5 l de 100x colorante fluorescente de intercalación) y 660 l (o 655 l) de agua a hacer 1 ml de tampón de reconstitución. Mezcle en un vórtex durante 10 s. </li><li> Utilice el tampón reconstituido preparado en la sección 6 para repetir los pasos 5.2 - 5.6. </li><li> Visualizar los resultados por el ojo desnudo (de reacción que contiene HNB) o una luz UV (de reacción que contiene el colorante intercalante fluorescente). </li></ol> 7. Realizar reacción LAMP en tiempo real con escáner Tubo <ol><li> Añadir 5 l de ADN para la reconstituted reactivos que contienen colorante intercalante fluorescente, cerca del tubo y la inserta en el escáner de tubo. </li><li> Ajuste la temperatura de incubación a 60 ° C. Medir la fluorescencia a 520 nm durante 60 min. </li></ol>

<ol>
	<li>Anderson, A., 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=Diagnosis+and+management+of+Q+fever+United+States,+2013:+recommendations+from+CDC+and+the+Q+Fever+Working+Group.">Diagnosis and management of Q fever United States, 2013: recommendations from CDC and the Q Fever Working Group.</a> MMWR Recomm. Rep. 62 (3), 1-30 (2013).</li><li>Rolain, J. M., Boulos, A., Mallet, M. N., Raoult, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+ratio+of+serum+doxycycline+concentration+to+MIC+and+rapid+decline+of+antibody+levels+during+treatment+of+Q+fever+endocarditis.">Correlation between ratio of serum doxycycline concentration to MIC and rapid decline of antibody levels during treatment of Q fever endocarditis.</a> Antimicrob. Agents and Chemother. 49 (7), 2673-2676 (2005).</li><li>Fournier, P. E., Raoult, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+PCR+and+serology+assays+for+early+diagnosis+of+acute+Q+fever.">Comparison of PCR and serology assays for early diagnosis of acute Q fever.</a> J. Clin. Microbiol. 41 (11), 5094-5098 (2003).</li><li>Schneeberger, P. M., 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=Real-time+PCR+with+serum+samples+is+indispensable+for+early+diagnosis+of+acute+Q+fever.">Real-time PCR with serum samples is indispensable for early diagnosis of acute Q fever.</a> Clin. Vaccine Immunol. 17 (2), 286-290 (2010).</li><li>Turra, M., Chang, G., Whybrow, D., Higgins, G., Qiao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+acute+Q+fever+by+PCR+on+sera+during+a+recent+outbreak+in+rural+South+Australia.">Diagnosis of acute Q fever by PCR on sera during a recent outbreak in rural South Australia.</a> Ann. N Y Acad. Sci. 1078, 566-569 (2006).</li><li>Notomi, T., 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=Loop-mediated+isothermal+amplification+of+DNA.">Loop-mediated isothermal amplification of DNA.</a> Nucleic Acids Res. 28 (12), (2000).</li><li>Paris, D. H., Blacksell, S. D., Newton, P. N., Day, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple,+rapid+and+sensitive+detection+of+Orientia+tsutsugamushi.+by+loo-isothermal+DNA+amplification.">Simple, rapid and sensitive detection of Orientia tsutsugamushi. by loo-isothermal DNA amplification.</a> Trans. R. Soc. Trop. Med. Hyg. 102 (12), 1239-1246 (2008).</li><li>Huber, 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=Loop-mediated+isothermal+amplification+assay+targeting+the+47-kDa+gene+of+Orientia+tsutsugamushi.:+A+rapid+and+sensitive+alternative+to+real-time+PCR.">Loop-mediated isothermal amplification assay targeting the 47-kDa gene of Orientia tsutsugamushi.: A rapid and sensitive alternative to real-time PCR.</a> J. Med. Microb. Diagn. 1 (112), (2012).</li><li>Pan, L., Zhang, L., Wang, G., Liu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+sensitive+detection+of+the+ompB+gene+of+spotted+fever+group+rickettsiae+by+loop-mediated+isothermal+amplification.">Rapid, sensitive detection of the ompB gene of spotted fever group rickettsiae by loop-mediated isothermal amplification.</a> BMC Infect. Dis. 12 (254), (2012).</li><li>Mori, Y., Nagamine, K., Tomita, N., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+loop-mediated+isothermal+amplification+reaction+by+turbidity+derived+from+magnesium+pyrophosphate+formation.">Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation.</a> Biochem. Biophys. Res. Commun. 289 (1), 150-154 (2001).</li><li>Qiao, Y. M., 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=Loop-mediated+isothermal+amplification+for+rapid+detection+of+Bacillus+anthracis.spores.">Loop-mediated isothermal amplification for rapid detection of Bacillus anthracis.spores.</a> Biotechnol. Lett. 29 (12), 1939-1946 (2007).</li><li>Tomita, N., Mori, Y., Kanda, H., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+(LAMP)+of+gene+sequences+and+simple+visual+detection+of+products.">Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products.</a> Nat. Protoc. 3 (5), 877-882 (2008).</li><li>Thekisoe, O. M., 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=Stability+of+Loop-Mediated+Isothermal+Amplification+(LAMP)+reagents+and+its+amplification+efficiency+on+crude+trypanosome+DNA+templates.">Stability of Loop-Mediated Isothermal Amplification (LAMP) reagents and its amplification efficiency on crude trypanosome DNA templates.</a> J. Vet. Med. Sci. 71 (4), 471-475 (2009).</li><li>Chen, H. W., Ching, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+loop-mediated+isothermal+amplification+assays+for+rapid+and+easy+detection+of+Coxiella+Burnetii.">Development of loop-mediated isothermal amplification assays for rapid and easy detection of Coxiella Burnetii.</a> J. Microbiol Methods. 107, 176-181 (2014).</li><li>Wang, D., 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=A+Comparison+of+In-House+Real-Time+LAMP+Assays+with+a+Commercial+Assay+for+the+Detection+of+Pathogenic+Bacteria.">A Comparison of In-House Real-Time LAMP Assays with a Commercial Assay for the Detection of Pathogenic Bacteria.</a> Molecules. 20 (6), 9487-9495 (2015).</li></ol>

Los autores no tienen nada que revelar.

Anteriormente, hemos desarrollado un ensayo de lámpara sensible y específico de orientación del elemento de inserción 14 IS1111a. El elemento IS1111 fue seleccionado porque es muy conservadas entre las diferentes cepas de C. burnetii y su alto número de copias (7-110) en la bacteria (Klee 2006). Nuestros resultados muestran que la lámpara puede detectar aproximadamente 25 copias del elemento IS1111, que puede correlacionarse con tan poco como una copia cromosómica de...

La pequeña bacteria Gram-negativa Coxiella burnetii es el agente causante de la fiebre Q, que es una zoonosis en todo el mundo. Debido a la distribución mundial de la fiebre Q y la alta infectividad de C. burnetii, personal militar y civil de Estados Unidos desplegadas en el extranjero están en riesgo de ser infectados en las áreas endémicas. La fiebre Q se manifiesta en dos formas: infecciones agudas y crónicas. La fiebre Q aguda se presenta con síntomas similares a la gripe, hepatitis, o la neumonía, y suele ser una enfermedad autolimitada con una baja tasa de mortalidad. Q-fiebre crónica, mientras que menos frecuente, a menudo resulta en endocarditis, que tiene una tasa de mortalidad mucho más alta si se deja sin tratamiento 1,2. Por lo tanto, el diagnóstico precoz para guiar un tratamiento adecuado es fundamental para el cuidado del paciente. Reacción en cadena de la polimerasa (PCR) y PCR cuantitativa en tiempo real (qPCR) se han desarrollado ensayos para la detección de C. ADN burnetii en cultivos celulares y muestras clínicas 3-5 &lt;/ Sup&gt;. Tanto PCR y qPCR son costosos ya menudo no es fácilmente disponible en las zonas con recursos limitados para el trabajo de rutina. Originalmente descrito por Notomi 6, amplificación isotérmica mediada por bucle (LAMP) ofrece un método de amplificación de ADN alternativo. Lámpara utiliza ADN polimerasa Bst para la síntesis de ADN de desplazamiento de cadena junto con conjuntos de cebadores especialmente diseñados que reconocen al menos seis regiones independientes del gen diana. La ventaja más significativa de LAMP es que la amplificación se produce en condiciones isotérmicas. Por lo tanto, sólo se requiere un baño de agua, calentador o una incubadora. Este método ha sido utilizado para detectar varios patógenos rickettsias 7-9. La visualización de los productos de ADN amplificados por electroforesis en gel es el método más preciso que puede diferenciar verdaderos positivos de falsos positivos debido a la amplificación no específica. Sin embargo, los procedimientos involucrados en la electroforesis en gel no son prácticos para ar de recursos limitadosEAS. Varios métodos alternativos han sido desarrollados para detectar los productos de reacción. Estos alternativa, como turbidez derivado de la formación de pirofosfato de magnesio 10 o el uso de un colorante intercalante fluorescente para ser visualizado con luz UV 11,12 son más favorables que la ejecución de un gel. Los reactivos de LAMP fueron estables durante un mes cuando se almacenaron a 25 ° C y 37 ° C, que son las temperaturas ambiente en los países tropicales y subtropicales donde la fiebre Q es endémica 13. Un ensayo de lámpara fue desarrollado en nuestro laboratorio para detectar la presencia de C. burnetii en muestras de plasma 14. Aquí se describe un protocolo sencillo para la preparación de la mezcla de reacción LAMP a partir de los reactivos liofilizados. Los reactivos liofilizados son estables durante un mes cuando se almacena a temperatura ambiente. Cuando se combina con un método de visualización fácil, esto es un método ideal para usar para la detección de C. burnetii en un entorno de recursos limitados.

<table border="0" cellpadding="0" cellspacing="0" width="773">
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">LAMP primers</td>
			<td style="width:188px;">Eurofins MWG operon</td>
			<td style="width:131px;">10 nmol, salt free</td>
			<td style="width:240px;">Sequence designed by customer</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Bst DNA polymerase</td>
			<td style="width:188px;">New England Biolabs</td>
			<td style="width:131px;">M0275L</td>
			<td style="width:240px;">8 units/ml</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">10X Thermo pol buffer</td>
			<td style="width:188px;">New England Biolabs</td>
			<td style="width:131px;">B9004S</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">dNTP mixture</td>
			<td style="width:188px;">New England Biolabs</td>
			<td style="width:131px;">N0447L</td>
			<td style="width:240px;">10 mM each</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Betaine</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:131px;">B0300</td>
			<td style="width:240px;">5 M, Trimethylglycine</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Magnesium Sulfate</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:131px;">M3409-1ML</td>
			<td style="width:240px;">1 M</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">10X Bluejuice</td>
			<td style="width:188px;">Invitrogen</td>
			<td style="width:131px;">10816-015</td>
			<td style="width:240px;">gel loading buffer</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">SYBR green</td>
			<td style="width:188px;">Invitrogen</td>
			<td style="width:131px;">S7585</td>
			<td style="width:240px;">10,000X, visualize products in tubes</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">GelRed</td>
			<td style="width:188px;">Phenix Research Products</td>
			<td style="width:131px;">RGB-4103</td>
			<td style="width:240px;">10,000X, visualize products in gels</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Lyophilized reagents</td>
			<td style="width:188px;">Gene Reach</td>
			<td style="width:131px;"></td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Hydroxynaphthol blue</td>
			<td style="width:188px;">Fluka</td>
			<td style="width:131px;">33936-10G</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">ESEQuant tube scanner</td>
			<td style="width:188px;">Qiagen</td>
			<td style="width:131px;"></td>
			<td style="width:240px;">real-time detection</td>
		</tr>
	</tbody>
</table>

the development of lyophilized loop-mediated isothermal amplification reagents for the detection of coxiella burnetii

Coxiella burnetii, el agente que causa la fiebre Q, es una bacteria intracelular obligado. Ensayos de diagnóstico basado en la PCR se han desarrollado para la detección de C. ADN burnetii en cultivos celulares y muestras clínicas. PCR requiere un equipo especializado y una amplia formación de usuario final, y por lo tanto, no es adecuado para el trabajo de rutina especialmente en un área de recursos limitados. Hemos desarrollado un ensayo de amplificación isotérmica mediada por bucle (LAMP) para detectar la presencia de C. burnetii en muestras de pacientes. Este método se realiza a una única temperatura de alrededor de 60 ° C en un baño de agua o un bloque de calentamiento. La sensibilidad de este ensayo LAMP es muy similar a la PCR con un límite de detección de aproximadamente 25 copias por reacción. Este informe describe la preparación de la reacción usando reactivos liofilizados y visualización de los resultados utilizando azul de hidroxinaftol (HNB) o una lámpara de UV con colorante intercalante fluorescente en la reacción. Los reactivos de LAMP fueron lyophilizeta y se almacena a temperatura ambiente (RT) durante un mes, sin pérdida de sensibilidad de detección. Este ensayo LAMP es particularmente robusto porque la preparación de la mezcla de reacción no implica pasos complejos. Este método es ideal para su uso en entornos con recursos limitados donde la fiebre Q es endémica.

Los reactivos liofilizados de LAMP en el interior del tubo de 0,2 ml contiene ADN polimerasa Bst, cebadores y dNTPs. 20 l de tampón de reconstitución se utiliza para volver a suspender los reactivos liofilizados. La figura 1 muestra los resultados de la reacción de lámpara en geles de agarosa con reactivos recién preparados y reactivos liofilizados. El reactivo liofilizado no reduce su actividad. LAMP reacciones preparados por ambos reactivos puede detectar...

Watch this Scientific Journal Video about The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii at JoVE.com

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

El desarrollo de la mediación de la Loop liofilizada amplificación isotérmica reactivos para la detección de<em&gt; Coxiella burnetii</em

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

the-development-lyophilized-loop-mediated-isothermal-amplification

Research

JoVE Journal

Immunology and Infection

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

10.4K vistas.  Naval Medical Research Center. We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Video: The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Detección del virus infeccioso de campo recogidos por los mosquitos del ensayo cultivo celular Vero

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Investigación

Inmunología e Infección

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

Generación de Arenavirus recombinante para el Desarrollo de Vacunas de la FDA aprobados células Vero

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

Evaluar el desarrollo de las células murino plasmocitoide dendríticas en las placas de Peyer Usando la transferencia adoptiva de Progenitores Hematopoyéticas

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

El análisis in vitro de la respuesta inmune celular Myd88 mediada por el Virus del Nilo Occidental cepa mutante Infección

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

Desarrollo de una<em&gt; In vitro</em&gt; Sistema modelo para estudiar la interacción de<em&gt; Equus</em&gt;<em&gt; Caballus</em&gt; IgE con su receptor de alta afinidad Fc RI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Isotérmico de amplificación (LAMP) Ensayos Loop mediada para la detección específica de las especies de<em&gt; Eimeria</em&gt; Que infectan pollos

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Generación y multi-fenotípicos cribado de alto contenido de<em&gt; Coxiella burnetii</em&gt; transposón mutantes

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

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 ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

La aplicación de fluorescencia la transferencia de energía de resonancia (FRET) para examinar la eficiencia de efector por translocación<em&gt; Coxiella burnetii</em&gt; Durante siRNA silenciamiento

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Desarrollo de una más sensible y específica cromogénico medio de agar para la detección de<em&gt; Vibrio parahaemolyticus</em&gt; Y Otros<em&gt; Vibrio</em&gt; Especies

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...