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2013, Número 2

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Biotecnol Apl 2013; 30 (2)


Evaluación de cuatro métodos de extracción de ARN viroide para el diagnóstico molecular de CEVd en Citrus limon mediante RT-PCR, Dot blot y Northern blot

Umaña R, Pritsch C, Arbiza JR, Rivas F, Pagliano G

Idioma: Español
Referencias bibliográficas: 39
Paginas: 125-136
Archivo PDF: 420.92 Kb.


RESUMEN

En el desarrollo de metodologías moleculares diagnósticas en variedades cítricas propensas a infección viroide, se precisa la extracción eficiente de ARN, siguiendo criterios de concentración y pureza. Los contaminantes remanentes pueden afectar la detección según la herramienta molecular escogida. Esta condición se analiza a partir del espectro de absorción del ARN; mediante la reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR) y por los niveles de intensidad de las señales de Northern blot y Dot blot, en términos de respuesta analítica/sensibilidad. Se evaluaron cuatro métodos de extracción de ARN, por sus efectos sobre la detección de presencia/ausencia del Citrus Exocortis Viroid (CEVd) en Citrus limon mediante análisis moleculares diagnósticos: 1) extracción viroide convencional (EVC); 2) fenol/tiocianato de guanidina (FTG); 3) SDS/acetato de potasio (SAP); y 4) formaldehído/SSC (FS). Los valores del tejido de floema estuvieron entre 7500 ng/µL y 1200 ng/µL y los rangos entre 1.3 y 2.0 DO260/280. La evaluación por aproximaciones de la RT-PCR reflejó las amplificaciones esperadas del genoma completo del CEVd; sin embargo, aún se discuten los escenarios erráticos. Las hibridaciones no radiactivas revelaron señales de alta intensidad (132 UR) para el tejido infectado, según el método de EVC, y la definición del límite de positividad para la presencia de infección (78 UR). Las herramientas basadas en hibridaciones moleculares interfieren en el diagnóstico, por la rigurosidad del protocolo y las condiciones del ARN molde. La extracción viroide como punto de partida de una detección exitosa y los métodos moleculares ensayados, mostraron las posibilidades diagnósticas de la asociación de Northern blot con la EVC.


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