Los autores están en deuda con los jefes, jefes y comunidades de Macha para su unwaveing ​​cooperar durante los mosquitos colecciones de campo de muestra. Dr. Doug Kent Norris y Rebeca ofreció invaluable experiencia en el protocolo de desalado. Este trabajo fue financiado por la Universidad Johns Hopkins Malaria Research Institute sistema piloto de subvención. Mosquitos y parásitos patrones de ADN de laboratorio fueron donados amablemente por MR4, American Type Culture Collection y.

1. Disección Preparatoria de muestras de mosquitos <ol><li> Lugar de mosquito en un pequeño corte de parafina y de montaje en microscopio de disección. </li><li> Cubrir en gota de agua desionizada para ablandar el tejido. </li><li> Incisión de la carcasa mosquito precisamente en la unión entre el tórax y el abdomen, por lo tanto mellar la cabeza y el tórax de la sección abdominal. </li><li> Transfiera cada sección mosquitos disecados en un lugar limpio autoclave 1,5 ml tubo de microcentrífuga prelabeled con mosquitos detalles de identificación de números y debidamente marcados para indicar la sección del cuerpo (por ejemplo, &quot;m&quot; para mediados de intestino sección; &quot;ht&quot; para la cabeza y el tórax sección). </li><li> Deseche parafilm y limpie cuidadosamente platina del microscopio con un 70% de alcohol humedecido Kimwipe antes de procesar el siguiente mosquito. </li></ol> 2. Extracción de ADN a partir de muestras de mosquitos <ol><li> Pipetear 20 l de agua desionizada en un tubo de muestra y la punta de pipeta de uso para moler la sección de mosquito sumergido en una ONUsuspensión iform. </li><li> Añadir 100 l de autoclave 1X PBS / 1% de solución de saponina al homogeneizado de la muestra y mezclar mediante agitación suave momentánea. </li><li> Incubar a temperatura ambiente durante 20 min. </li><li> Centrifugar a 20.000 xg durante 2 min y desechar el sobrenadante. </li><li> Resuspender el sedimento en 100 l de 1X PBS. </li><li> Centrifugar de nuevo a 20.000 xg durante 2 min y desechar el sobrenadante. </li><li> Por agitación suave (5 segundos), se resuspende el precipitado en 75 l de agua desionizada estéril y 25 l de w / v 20% de suspensión de resina Chelex-100 en agua desionizada. </li><li> Pierce agujero en la tapa del tubo de muestra con aguja hipodérmica estéril 23G flameado en el quemador Bunsen. </li><li> Hervir suspensión de la muestra en un baño de agua sobre una rejilla flotante (o en bloque de calentamiento) durante 10 min. </li><li> Centrifugar a 20.000 xg, durante 1 min y la transferencia subsiguiente solución de ADN en el vial de almacenamiento prelabeled para su uso como plantilla en aplicaciones de la PCR. </li></ol> 3. La genotipificación Plasmodium falciparum y Anophelesspp. Identificación molecular <ol><li> Añadir 2,5 l de extracto de ADN Chelex en 25 mu l reacciones de PCR para comprobar la calidad del ADN 11, identificar las especies de Anopheles 10 y el genotipo P. falciparum 12 del intestino medio y salival infecciones de la glándula. </li><li> Para maximizar el rendimiento del amplicón, especialmente para P. detección falciparum, 1,5 X incluyen albúmina de suero bovino (BSA) en el ensayo de PCR. La composición de reacción se recomienda la siguiente: (2,5 l plantilla, 0,25 cebadores mu M, cloruro de magnesio 1,5 mM, dNTPs 200 mu M, 1X tampón de PCR, BSA 1,5 X y 1.0U Taq ADN polimerasa, en 25 volúmenes mu l). </li><li> Para comprobar la calidad del ADN en extracto, amplificar la región de la deshidrogenasa mitocondrial de artrópodos dinucleótido de nicotinamida adenina (NADH) subunidad 4 (ND4) gen (ND4FW cebadores [5&#39;-GTD YAT TTA TGA CCT TTR AA-3 &#39;] y ND4RV [5&#39; -CTT CTT CAC EGC ADW CGT TC-3 &#39;]; esperan que el producto 400 pb) 11,13. </li><li> Para diferenciar Anopheles gambiae sl. especies hermanas (An. gambiae ss y An. arabiensis. solamente), amplificar región de acompañamiento SNPs en el espaciadoras intergénicas (IGS) región (primers de la ONU [5&#39;-GTGTGCCCCTTCCTCGATGT-3 &#39;], GA [5&#39;-CTGGTTTGGTCGGCACGTTT- 3 &#39;], AR [5&#39;-AAGTGTCCTTCTCCATCCTA-3&#39;]; esperar 390bp productos An. SS gambiae, 315bp An. arabiensis) 10,11. </li><li> Para escribir P. falciparum antifolato polimorfismos de resistencia a fármacos, realizar la PCR anidada para amplificar la región flanqueante codones de aminoácidos 108, 51, 59, 16 y 164 en la dihidrofolato reductasa parásito (DHFR): Cebadores primarios redondas: M1 [5&#39;-TTTATGATGGAACAAGTCTGC-3 &#39;] y M5 [5&#39;-AGTATATACATCGCTAACAGA-3&#39;] Secondary cebadores redondas: M3 [5&#39;-TTTATGATGGAACAAGTCTGCGACGTT-3 &#39;] con F / [5&#39;-AAATTCTTGATAAACAACGGAAACCTTTTA-3&#39;] O F [5&#39;-GAAATGTAATTCCCTAGATATGGAATATT-3] con M4 [5&#39;-TTAATTTCCCAAGTAAAACTATTAGAGCTTC-3 &#39;]). También amplificar la región que contiene los codones de aminoácidos 436, 437, 540, 581 aº 613 en la P. falciparum dihidropteroato sintetasa (DHPS) de genes: Primaria ronda primers R2 [5&#39;-AACCTAAACGTGCTGTTCAA-3 &#39;] con R / [5&#39;-AATTGTGTGATTTGTCCACAA3&#39;]; Secundaria primers redondo J: [5&#39;-&#39;TGCTAGTGTTATAGATATAGGTGGAGAAAGC-3&#39;] con K / [5&#39;-CTATAACGAGGTATTGCATTTATTGCAAGAA-3 &#39;] O K: [5&#39;-TGCTAGTGTTATAGATATAGGATGAGCATC-3 &#39;] con K /, O L [5&#39;-ATAGGATACTATTTGATATTGATATTGGACCAGGATTCG-3 &#39;] con L / [5&#39;-5TATTACAACATTTTGATCATTCGCGCAACCGG-3&#39;] 12. </li><li> Tema 4 amplicón l a alelo-específicos digestiones de enzimas de restricción en 30 reacciones mu l siguiendo las instrucciones del fabricante, para detectar fármacos antifolato asociadas a la resistencia 12 polimorfismos. </li><li> Tema 5 l de amplificación de PCR (o 15 l de digestión de restricción) por carril de etidio teñido con bromuro de 2% en gel de agarosa para electroforesis (100 - 120V) y visualizar las bandas con transiluminación UV. </li></ol>

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No hay conflictos de interés declarado.

El método de Chelex simplificada presentada aquí permite la extracción de la calidad Anopheles spp y P. falciparum ADN de las muestras de mosquitos susceptibles de diversas aplicaciones de la PCR. Esta técnica se puede emplear para la identificación molecular de los mosquitos vectores de la malaria y la vigilancia de resistente a los medicamentos P. alelos falciparum en los mosquitos para los programas nacionales de control de la malaria. Las ventajas de la técnica de Chelex simplificado incluyen la simplicidad, menos reactivos y, por tanto costo, seguridad y tiempo de procesamiento más corto (37 min) que los protocolos estándar tales como el método de precipitación salina 10 (2 hr 47 min y una etapa durante la noche, Tabla 1). Las ventajas antes mencionadas y los requisitos mínimos de reactivo (3 reactivos) en comparación con el actual protocolo estándar (10 reactivos, Tabla 1) 11,15, que el protocolo Chelex simplificado particularmente amigable para los laboratorios del país endémicas donde un reactivo constantecadena de suministro es a menudo difícil de mantener. Una limitación de este método es que como el protocolo estándar, que fue también objeto de inhibidores de la PCR sabe que se producen en el integumento mosquito 16. Sin embargo, esto es fácilmente aliviado por la inclusión de BSA en los ensayos. BSA también se ha empleado con éxito como un potenciador de la amplificación en contra de inhibidores en otras aplicaciones de la PCR 17,18.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Chelex-100, 50-100 mesh seco </td><td> BioRad </td><td> # 143-2832 </td><td></td></tr><tr><td> La saponina </td><td> SIGMA </td><td> 142-7425 </td><td></td></tr><tr><td> BSA </td><td> New England Biolabs </td><td> B9001S </td><td></td></tr></tbody></table>

a simple chelex protocol for dna extraction from anopheles spp.

Los países endémicos están adoptando cada vez más herramientas moleculares para la tipificación eficiente, identificación y vigilancia contra parásitos de la malaria y los mosquitos vectores, como parte integral de sus programas de control de 1,2,3,4,5. Para el establecimiento sostenible de estos enfoques precisos en las operaciones de investigación para fortalecer el control de la malaria y los esfuerzos de eliminación, métodos sencillos y asequibles, con el reactivo parsimonioso y requerimientos de equipo son esenciales 6,7,8. Aquí les presentamos una sencilla técnica basada en Chelex para la extracción de parásito de la malaria y el ADN vector de muestras de campo mosquitos recolectados. Hemos identificado morfológicamente 72 Anopheles gambiae sl. Desde 156 mosquitos capturados por piretro spray de capturas en los dormitorios de los hogares dentro de 2.000 km 2 cerca del Instituto de Malaria en Macha. Después de la disección para separar la cabeza y el tórax del abdomen para todos los gam 72 AnophelesBIAE sl. mosquitos, las dos secciones se colocaron individualmente en tubos de 1,5 ml de microcentrífuga y se sumergieron en 20 l de agua desionizada. Utilizando una punta de pipeta estéril, cada sección de mosquito se homogeneizó por separado a una suspensión uniforme en el agua desionizada. Del homogeneizado resultante de cada sección de mosquito, 10 l se retuvo mientras que el otro 10 l se transfirió a un tubo esterilizado en autoclave por separado 1,5 ml. Las alícuotas separadas se sometieron a extracción de ADN ya sea por el Chelex simplificado estándar o la salazón a cabo la extracción protocolo de 9,10. El protocolo de salificación es llamado y utilizado ampliamente debido a que emplea altas concentraciones de sal en lugar de disolventes orgánicos peligrosos (tales como fenol y cloroformo) para la etapa de precipitación de la proteína durante la extracción de ADN 9. Los extractos fueron utilizados como plantillas para la amplificación por PCR usando cebadores dirigidos artrópodo deshidrogenasa mitocondrial nicotinamida adenina dinucleótidonase (NADH) subunidad gen 4 (ND4) para comprobar la calidad del ADN 11, una PCR para la identificación de las especies de Anopheles gambiae hermanos de 10 y una PCR anidada para la tipificación de infección por Plasmodium falciparum 12. La comparación con la calidad del ADN (ND4) PCR mostró 93% de sensibilidad y 82% especificidad para el enfoque Chelex en relación con lo establecido desalado protocolo. Los valores correspondientes de sensibilidad y especificidad fueron del 100% y 78%, respectivamente, utilizando hermano de identificación de especies PCR y 92% y 80%, respectivamente, para P. falciparum detección por PCR. No hubo diferencias significativas en la proporción de muestras en las que la señal de amplicón con el Chelex o el desplazamiento salino normal protocolo en las tres aplicaciones de la PCR. El enfoque Chelex requiere tres reactivos simples y 37 min en completarse, mientras que el. Desalado protocolo implicó 10 diferentes reactivos y 2 h 47 min y el tiempo de «tratamiento, incluyendo una etapa durante la noche Nuestros resultados muestran then el método de Chelex es comparable a la existente salazón a cabo la extracción y pueden estar sustituidos como un enfoque sencillo y sostenible en entornos con recursos limitados donde una cadena de suministro constante reactivo a menudo es difícil de mantener.

Los ejemplos de los resultados de los ensayos de PCR para la calidad del mosquito extracto de ADN (Figura 1), Anopheles arabiensis identificación molecular (Figura 2) y P. falciparum de detección (Figura 3) muestran que el método de Chelex simplificado produce resultados similares a la norma salificación protocolo 10, a pesar de los pasos mucho menos (Tabla 1). Con una calidad de ADN comparable en los extractos respectivos, no es sorprendente que las tasas de muestra positividad con respecto a una. gambiae especies de hermanos, así como las tasas de infección por el parásito no fue estadísticamente diferente en función de chi-cuadrado de McNemar prueba (Figura 3). Sensibilidad (%) se calculó como TP / (TP + FN) * 100, donde TP indica verdaderos positivos y falsos negativos FN marca. Especificidad (%) se determinó como TN / (TN + FP) * 100, donde TN indica un resultado negativo y FP denota falsos positivos. La calidad del ADN (ND4) PCR mostró 93% de sensibilidad y 82% especificidad para el enfoque Chelex en comparación con el protocolo establecido desalado como estándar de oro. Los valores correspondientes de sensibilidad y especificidad fueron del 100% y 78%, respectivamente, utilizando hermano de identificación de especies PCR y 92% y 80%, respectivamente, para P. falciparum detección por PCR. La adición de BSA a las mezclas de reacción dio como resultado un aumento general de positivos de PCR (Figura 3) debido al alivio de los inhibidores de la PCR 14, tanto para el Chelex simplificado y regular salificación protocolo. Sin embargo, este aumento no fue estadísticamente significativo, excepto para la calidad del ADN (PCR ND4) en extractos de Chelex (p = 0,039). El alelo-específica restricción de la digestión enzimática de la P. falciparum DHFR (o gen diana otro) amplicón permite el genotipado de las infecciones de malaria del intestino medio y de las glándulas salivales para los alelos de resistencia a fármacos (Figura 4, los datos de las glándulas salivales no se muestra). <table border = &quot;1&quot;&gt; <tbody><tr><td colspan="2"> Procedimiento simple Chelex </td><td colspan="2"> Estándar Ensanchamiento Procedimiento </td></tr><tr><td> Pasos </td><td> Reactivos </td><td> Pasos </td><td> Reactivos </td></tr><tr><td><ol><li> Añadir la muestra a 1,5 ml tubo de microcentrífuga y se homogeneiza en 100 l de 1X PBS / 1% de solución de saponina (2 min). </li><li> Dejar a temperatura ambiente durante 20 min. </li><li> Girar a 20.000 xg durante 2 min y desechar el sobrenadante. </li><li> Añadir 100 l de PBS 1X y mezclar suavemente por agitación momentánea (3 seg.) </li><li> Girar a 20.000 xg durante 2 min y desechar el sobrenadante. </li><li> Añadir 25 l de w / v Chelex 20% en agua desionizada y 75 l de agua desionizada estéril. </li><li> Pierce agujero en la tapa del tubo de muestra usando una aguja caliente estéril hipodérmica 23G y el contenido del tubo ebullición durante 10 min. </li><li> Derivado tubo de microcentrífuga a 20.000 xg durante 1 min. </li><li> Transferir el sobrenadante en un vial estéril prelabeled y solución de ADN almacenar a -20 ° C hasta su uso (2,5 l en 25 l de reacción de PCR). </li></ol></td><td> PBS La saponina Chelex-100 perlas </td><td><ol><li> Añadir la muestra a 1,5 ml tubo de microcentrífuga y se homogeneiza en 100 * l tampón Bender con 1% DEPC (2 min). </li><li> Incubar a 65 ° C durante 1 hr. </li><li> Añadir 15 l de acetato de potasio frío 8 M. Mezclar suavemente e incubar en hielo durante 45 min. </li><li> Derivado de muestra en tubos de microcentrífuga a 20.000 xg durante 10 min y Transferir el sobrenadante a un tubo nuevo 1,5 ml. </li><li> Añadir 250 l de etanol al 100% y mezclar bien invirtiendo el tubo. </li><li> Incubar la muestra a temperatura ambiente durante 5 min. </li><li> Girar las muestras a 20.000 xg durante 15 min. </li><li> Aspirar y desechar el sobrenadante, dejando el precipitado se seque completamente en el tubo (30 men). </li><li> Resuspender el sedimento en 20 l de 0,1 x SSC + RNAsa (10 mg / ml) durante la noche a 4 ° C. </li><li> Añadir 80 l de agua DEPC y se almacena a -20 ° C hasta su uso. </li></ol></td><td> Dietilpirocarbonato (DEPC) * Bender Buffer: 0,1 M NaCl 0,2 M de sacarosa 0,1 M Tris-HCL 0,05 M EDTA, pH 9,1 0,5% de SDS en agua DEPC 8 M de acetato de potasio Etanol 0,1 X citrato salino sódico (SSC) de amortiguación RNAsa (10 mg / ml) </td></tr><tr><td colspan="2"> TIEMPO TOTAL: 37 min </td><td colspan="2"> Tiempo total: 2 horas 47 minutos, y durante la noche </td></tr></tbody></table> Tabla 1. Paso a paso comparación de los reactivos y el tiempo requerido para el protocolo Chelex simple y soportarard desalado método de extracción de ADN a partir de muestras de mosquitos. <img alt="Figura 1" src="/files/ftp_upload/3281/3281fig1.jpg" /> Figura 1. ND4 mitocondrial PCR comparación de la calidad del ADN de Chelex simplificado &quot;C&quot; y estándar de precipitación salina &quot;M&quot; extractos de las muestras de Anopheles arabiensis de campo. NC, control negativo; M1 y C1, C2 y M2, M3 y C3, y C4 y M4 están emparejados desalado y extractos Chelex de un mismo. especímenes arabiensis mosquitos. L, escalera de 100 pb de ADN. <img alt="Figura 2" src="/files/ftp_upload/3281/3281fig2.jpg" /> Figura 2 Molecular PCR para la identificación Anopheles arabiensis C1 y M1, M2 y C2, C3 y M3, M4 y C4 denotar amplicón correspondiente de la salazón a cabo &quot;M&quot; y simplificado Chelex &quot;C&quot; extractos de ADN de las muestras de mosquitos mismos;.. AR, Anopheles arabiensiscontrol positivo, L, escalera de 100 pb de ADN. <img alt="Figura 3" src="/files/ftp_upload/3281/3281fig3.jpg" /> Figura 3. Detección de positivos en Chelex y desalado extractos de ADN de muestras de campo de mosquitos, con los ensayos de PCR para la identificación molecular de Anopheles arabiensis (AR) 10, artrópodos genes NADH deshidrogenasa 4 (ND4) Prueba de ADN de calidad 11, y P antifolato resistencia a los medicamentos . falciparum DHFR genotipado 12 (F-M4). Los resultados mostrados para los ensayos se ejecutan con o sin BSA en la mezcla de reacción. Chi-cuadrado de McNemar se utilizó la prueba para determinar si las diferencias entre ciento de PCR positiva de ADN extraído de la Chelex simplificado y el estándar de precipitación salina protocolos fueron estadísticamente significativas. <img alt="Figura 4" src="/files/ftp_upload/3281/3281fig4.jpg" /> <strong&gt; Figura 4. Aplicación del protocolo simplificado Chelex en genotipificación P. infecciones por P. falciparum en los mosquitos (intestino medio muestran los datos) para los alelos de resistencia a fármacos. BstN I digestión de amplificación que flanquea el codón 108 del P. falciparum gen DHFR 12 muestra cicloguanil mutantes resistentes S108T en muestras de mosquitos (M1-M5; del intestino medio se muestran los datos). U, sin digerir 522 pb de amplificación; FCR3, laboratorio estándar P. Falciparum clon control positivo llevar S108T, K1, P. falciparum laboratorio estándar de control clon negativo que lleva cicloguanil sensible S108N, L, escalera de 100 pb de ADN.

Watch this Scientific Journal Video about A Simple Chelex Protocol for DNA Extraction from Anopheles spp. at JoVE.com

A Simple Chelex Protocol for DNA Extraction from Anopheles spp. | Immunology and Infection | Video

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Una forma rápida y asequible para extraer parásito calidad malaria y ADN del vector a partir de muestras de mosquitos se describe. Capitalizando en las propiedades quelantes de resina Chelex, el simple método permite el genotipado de parásitos de la malaria en mosquitos fases glándula del intestino medio y salivales, así como la identificación molecular de los<em&gt; Anopheles</em&gt; Entre hermanos especies por PCR.

Un protocolo Chelex simple para la extracción de ADN a partir de<em&gt; Anopheles</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

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A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

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54.0K vistas.  Malaria Institute at Macha. Una forma rápida y asequible para extraer parásito calidad malaria y ADN del vector a partir de muestras de mosquitos se describe. Capitalizando en las propiedades quelantes de resina Chelex, el simple método permite el genotipado de parásitos de la malaria en mosquitos fases glándula del intestino medio y salivales, así como la identificación molecular de los Anopheles Entre hermanos especies por PCR.

Video: A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

Un protocolo para la recolección y la tinción de los hemocitos del mosquito de la fiebre amarilla Aedes aegypti</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

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Inmunología e Infección

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

Hens can be immunized by means of i.m. vaccination (Musculus pectoralis, left and right, injection volume 0.5-1.0 ml) or by means of Gene-Gun plasmid-immunization. Dependent on the immunogenicity of the antigen, high antibody-titres (up to 1:100,000 - 1:1,000,000) can be achieved after only one or 3 - 4 boost immunizations. Normally, a hen lays eggs continuously for about 72 weeks, thereafter the laying capacity decreases. This protocol describes the extraction of total IgY from egg yolk by means of a precipitation procedure (PEG. Polson et al. 1980). The method involves two important steps. The first one is the removal of lipids and the second is the precipitation of total IgY from the supernatant of step one. After dialysis against a buffer (normally PBS) the IgY-extract can be stored at -20&deg;C for more than a year. The purity of the extract is around 80 %, the total IgY per egg varies from 40-80 mg, dependent on the age of the laying hen. The total IgY content increases with the age of the hen from around 40 mg/egg up to 80 mg/egg (concerning PEG precipitation). The laying capacity of a hen per year is around 325 eggs. That means a total potential harvest of 20 g total IgY/year based on a mean IgY content of 60 mg total IgY/egg (see Table 1).

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

This protocol describes in particular the extraction of total IgY from egg yolk by means of polyethylene glycol precipitation and gives general information about IgY technology.

Tecnología IgY: Extracción de anticuerpos de pollo de yema de huevo por el polietilenglicol (PEG) Precipitación

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important plant parasitic nematodes. The microscopic second-stage juvenile (J2), molted once in the egg, is the infective stage. The J2s hatch from the eggs, move freely in the soil within a film of water, and locate root tips of suitable plant species. After penetrating the plant root, they migrate towards the vascular cylinder where they establish a feeding site and initiate feeding using their stylets. The multicellular feeding site is comprised of several enlarged multinuclear cells called 'giant cells' which are formed from cells that underwent karyokinesis (repeated mitosis) without cytokinesis. Neighboring pericycle cells divide and enlarge in size giving rise to a typical gall or root knot, the characteristic symptom of root-knot nematode infection. Once feeding is initiated, J2s become sedentary and undergo three additional molts to become adults. The adult female lays 150-250 eggs in a gelatinous matrix on or below the surface of the root. From the eggs new infective J2s hatch and start a new cycle. The root-knot nematode life cycle is completed in 4-6 weeks at 26-28&deg;C.
Here we present the traditional protocol to infect plants, grown in pots, with root-knot nematodes and two methods for high-throughput assays. The first high-throughput method is used for plants with small seeds such as tomato while the second is for plants with large seeds such as cowpea and common bean. Large seeds support extended seedling growth with minimal nutrient supplement. The first high throughput assay utilizes seedlings grown in sand in trays while in the second assay plants are grown in pouches in the absence of soil. The seedling growth pouch is made of a 15.5 x 12.5cm paper wick, folded at the top to form a 2-cm-deep trough in which the seed or seedling is placed. The paper wick is contained inside a transparent plastic pouch. These growth pouches allow direct observation of nematode infection symptoms, galling of roots and egg mass production, under the surface of a transparent pouch. Both methods allow the use of the screened plants, after phenotyping, for crossing or seed production. An additional advantage of the use of growth pouches is the small space requirement because pouches are stored in plastic hanging folders arranged in racks.

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Two distinct methods to screen plants with root-knot nematodes are described. The described approaches include high-throughput screens with nematodes in a nondestructive manner facilitating the use of these plants in breeding programs.

Alto y Bajo Rendimiento pantallas con nematodos- Meloidogyne spp.</em

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important ...

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

Un protocolo simple para el agrupamiento de plaquetas mediada por<em&gt; Plasmodium falciparum</emEritrocitos&gt; por el VIH en un recurso pobre ajuste

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

Un protocolo simple y rápida para no enzimáticamente disociarse Tejidos Humanos frescas para el Análisis de los linfocitos infiltrantes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Un enfoque simple y eficiente de construcción de mutantes de virus vaccinia Vectores

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

La combinación de análisis de la DNA en una extracción de crudo del virión con el análisis de ARN de hojas infectadas para descubrir nuevos genomas de Virus

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Extracción rápido, seguro y sencillo Manual cabecera de ácido nucleico para la detección del Virus en muestras de sangre entera

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Una plataforma de alto rendimiento para la detección de Salmonella spp. /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Analysis of Fecal Microbiota Dynamics in Lupus-Prone Mice Using a Simple, Cost-Effective DNA Isolation Method

Gut microbiota has an important role in educating the immune system. This relationship is extremely important for understanding autoimmune diseases that are not only driven by genetic factors, but also environmental factors that can trigger the onset and/or worsen the disease course. A previously published study on the dynamics of the gut microbiota in lupus-prone MRL/lpr female mice showed how changes of the gut microbiota can alter disease progression. Here, a protocol is described for extracting representative samples from the gut microbiota for studies of autoimmunity. Microbiota samples are collected from the anus and processed, from which the DNA is extracted using a phenol-chloroform method and purified by alcohol precipitation. After PCR is performed, purified amplicons are sequenced using a Next Generation Sequencing platform at Argonne National Laboratory. Finally, the 16S ribosomal RNA gene sequencing data is analyzed. As an example, data obtained from gut microbiota comparisons of MRL/lpr mice with or without CX3CR1 are shown. Results showed significant differences in genera containing pathogenic bacteria such as those in the phylum Proteobacteria, as well as the genus Bifidobacterium, which is considered part of the healthy commensal microbiota. In summary, this simple, cost-effective DNA isolation method is reliable and can help the investigation of gut microbiota changes associated with autoimmune diseases.

This protocol provides a simple, cost-effective DNA isolation method for the analysis of murine gut microbiota alterations during the development of autoimmune disease.

Análisis de la dinámica de la microbiota fecal en ratones propensos al lupus utilizando un método de aislamiento de ADN simple y rentable

Gut microbiota has an important role in educating the immune system. This relationship is extremely important for understanding autoimmune diseases that ...