Evaluación de la técnica de amplificación por recombinasa y polimerasa (RPA) para la detección de begomovirus presentes en cultivos de soja y poroto en Argentina
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Los métodos tradicionales de diagnóstico son imprecisos para la identificación de begomovirus. Actualmente se usan técnicas moleculares, que requieren equipamientos sofisticados o procedimientos complejos. La técnica de amplificación mediante recombinasa y polimerasa (RPA) es similar a la reacción en cadena de la polimerasa (PCR), sensible, específica, pero opera a temperatura constante. Con el fin de evaluar la utilización de esta técnica para la detección de begomovirus presentes en soja y poroto en Argentina, se diseñaron iniciadores específicos. Se probaron inicialmente por PCR, con clones de virus detectados en el país, y se logró amplificar una banda de 371pb. La RPA se llevó a cabo con el Twist Amp® Basic kit utilizando muestras de soja y poroto, sanas y enfermas y malezas infectadas. Se probaron dos métodos de conservación de muestras: hojas liofilizadas y hojas mantenidas a -70 ºC; y dos de obtención de ADN: CTAB y molido de la muestra en 0,5M OHNa. La reacción se incubó a 37 ºC durante 30 min, y luego a 65 ºC durante 10 min. Se visualizaron bandas del tamaño esperado en plantas infectadas y no en testigos sanos. No hubo diferencias según los métodos de conservación de material ni con los de extracción de ADN utilizados. Se logró ajustar la RPA para la detección de begomovirus en cultivos de soja y poroto de Argentina.
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