Evaluation of the RPA technique for the detection of begomoviruses present in soybean and bean crops in Argentina
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...
| Main Authors: | , |
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| Format: | Online |
| Language: | spa |
| Published: |
Facultad de Ciencias Agropecuarias
2018
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| Online Access: | https://revistas.unc.edu.ar/index.php/agris/article/view/19319 |
| _version_ | 1841853969579638784 |
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| author | Reyna, Pablo Gastón Rodriguez Pardina, Patricia Elsa |
| author_facet | Reyna, Pablo Gastón Rodriguez Pardina, Patricia Elsa |
| author_sort | Reyna, Pablo Gastón |
| collection | Portal de Revistas |
| description | 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. |
| format | Online |
| id | oai:ojs.revistas.unc.edu.ar:article-19319 |
| institution | Universidad Nacional de Cordoba |
| language | spa |
| publishDate | 2018 |
| publisher | Facultad de Ciencias Agropecuarias |
| record_format | ojs |
| spelling | oai:ojs.revistas.unc.edu.ar:article-193192020-03-30T18:36:48Z Evaluation of the RPA technique for the detection of begomoviruses present in soybean and bean crops in Argentina 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 Reyna, Pablo Gastón Rodriguez Pardina, Patricia Elsa Glycine max Phaseolus vulgaris amplificación mediante polimerasa recombinasa. Glycine max Phaseolus vulgaris Recombinase Polymerase Amplification. 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. 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. Facultad de Ciencias Agropecuarias 2018-12-27 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion application/pdf text/html https://revistas.unc.edu.ar/index.php/agris/article/view/19319 10.31047/1668.298x.v35.n2.19319 AgriScientia; Vol. 35 No. 2 (2018); 35-42 AgriScientia; Vol. 35 Núm. 2 (2018); 35-42 1668-298X 10.31047/1668.298x.v35.n2 spa https://revistas.unc.edu.ar/index.php/agris/article/view/19319/22673 https://revistas.unc.edu.ar/index.php/agris/article/view/19319/29296 |
| spellingShingle | Glycine max Phaseolus vulgaris amplificación mediante polimerasa recombinasa. Glycine max Phaseolus vulgaris Recombinase Polymerase Amplification. Reyna, Pablo Gastón Rodriguez Pardina, Patricia Elsa Evaluation of the RPA technique for the detection of begomoviruses present in soybean and bean crops in Argentina |
| title | Evaluation of the RPA technique for the detection of begomoviruses present in soybean and bean crops in Argentina |
| title_alt | 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 |
| title_full | Evaluation of the RPA technique for the detection of begomoviruses present in soybean and bean crops in Argentina |
| title_fullStr | Evaluation of the RPA technique for the detection of begomoviruses present in soybean and bean crops in Argentina |
| title_full_unstemmed | Evaluation of the RPA technique for the detection of begomoviruses present in soybean and bean crops in Argentina |
| title_short | Evaluation of the RPA technique for the detection of begomoviruses present in soybean and bean crops in Argentina |
| title_sort | evaluation of the rpa technique for the detection of begomoviruses present in soybean and bean crops in argentina |
| topic | Glycine max Phaseolus vulgaris amplificación mediante polimerasa recombinasa. Glycine max Phaseolus vulgaris Recombinase Polymerase Amplification. |
| topic_facet | Glycine max Phaseolus vulgaris amplificación mediante polimerasa recombinasa. Glycine max Phaseolus vulgaris Recombinase Polymerase Amplification. |
| url | https://revistas.unc.edu.ar/index.php/agris/article/view/19319 |
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