Evaluación de la hibridación de ácidos nucleicos no radiactivo dot-blot como herramienta de detección para citrus leaf blotch virus en Cuba

Lester Hernández-Rodríguez, Gabriel García-García, Juana María Pérez-Castro, Lochy Batista-Le Riverend, Victoria Zamora-Rodríguez, Inés Peña-Barzaga, Pedro Luis Ramos-González

Resumen

Se evaluó un método de hibridación de ácidos nucleicos no radioactivo en formato dot-blot para la detección precisa de citrus leaf blotch virus (CLBV: Citrivirus) en extractos de ARN totales de árboles de cítricos. Los fragmentos de ADN que conforman parcialmente los marcos de lectura abiertos que codifican la polimerasa (RdRp), la proteína de movimiento (MP) y la proteína de la cubierta (CP) de CLBV se utilizaron para generar sondas marcadas con digoxigenina (DIG-11-dUTP) mediante PCR. Para generar las sondas se utilizaron fragmentos de ADN clonados a partir del aislado de CLBV de Cuba. Se evaluaron varias condiciones de trabajo que incluyeron el uso de diferentes membranas, soluciones de hibridación y tampones de lavado. Con el método evaluado fue posible detectar hasta 40 pg de un plasmidio que contiene el genoma completo de CLBV, así como material genómico del virus en extractos de ARN de plantas de kumquat 'Nagami' (Fortunella margarita) y kumquat 'Round' (F. japonica) infectadas de forma natural con el virus. Los resultados evidenciaron que el método desarrollado es una herramienta útil y altamente recomendable, una vez sea validado, para su uso en los programas de saneamiento, cuarentena y certificación del material de propagación de cítricos en Cuba.

Palabras clave

certificación; CLBV; Citrivirus; Flexiviridae; diagnóstico; Fortunella; material de propagación

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