Evaluation of the RPA technique for the detection of begomoviruses present in soybean and bean crops in Argentina
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Abstract
Traditional diagnostic methods are non-specific for begomovirus identification. At present molecular techniques are used, which require sophisticated equipment or complex procedures. The recombinase polymerase amplification technique (RPA) is similar to the polymerase chain reaction (PCR), sensitive and specific, but operates at constant temperature. In order to evaluate the use of this technique for the detection of begomovirus present in soybeans and beans in Argentina, specific primers were designed. They were initially tested by PCR, using clones of the different begomoviruses detected in our country and a 371 pb band was successfully amplified. RPA was carried out using the Twist AMP ® Basic Kitto test soybean, bean and weed infected samples, as well as healthy soybean and bean controls. Two conservation methods were tested: lyophilized leaves and leaves maintained at -70 ºC; and two DNA extraction methods: CTAB and crude extract grounded in OH Na 0.5M. The reaction was incubated at 37 ºC / 30 min. and at 65 ºC / 10 min. Bands of the expected size were visualized in infected samples, and not in healthy controls. There were no differences in the results due to either method of conservation or DNA extraction. RPA technique was successfully optimized for the detection of begomoviruses infecting soybean and bean crops of Argentina.
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