Standardization and validation of the nested pcr test for the diagnosis of the genus xyleborus species (coleoptera: curculionidae: scolytinae)
. In this study it was standardized and validated the nested PCR test for rapid, sensitive and reliable detection of species of the genus Xyleborus using external primers CI-J-2183 and TL2-N-3014 and internal primers J2210 and N2739 that amplify a band of 500 bp in the region of the gene mitochondri...
| Główni autorzy: | , , , |
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| Format: | Online |
| Język: | spa |
| Wydane: |
Instituto de Ecología, A.C.
2017
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| Dostęp online: | https://azm.ojs.inecol.mx/index.php/azm/article/view/1008 |
| _version_ | 1799770145410777088 |
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| author | Sosa-Castillo, María Elena Lara Reyna, Joel Ortega Arenas, Laura Delia Judith Hernández, Alfonsina |
| author_facet | Sosa-Castillo, María Elena Lara Reyna, Joel Ortega Arenas, Laura Delia Judith Hernández, Alfonsina |
| author_sort | Sosa-Castillo, María Elena |
| collection | AZM |
| description | . In this study it was standardized and validated the nested PCR test for rapid, sensitive and reliable detection of species of the genus Xyleborus using external primers CI-J-2183 and TL2-N-3014 and internal primers J2210 and N2739 that amplify a band of 500 bp in the region of the gene mitochondrial Cytochrome Oxidase subunit 1(CO1). Also, DNA extraction from 26 specimens of Xyleborus was realized with kit Qiagen DNeasy ® mericom Food (DMF), not previously reported its use for their application in insects, which resulted in enough DNA and high-quality for amplification by PCR reactions. The method allowed to process a single insect by extraction, and obtain genetic material from specimens preserved in alcohol of up to eight years old. The detection limit was defined up to a concentration of 780 pg/uL. The PCR was optimized in a final volume of 15 µL without compromising quality of amplification. The standardized test allowed quality DNA, which ensured high reproducibility and sensitivity in the detection of species of Xyleborus and partial sequencing of the CO1 gene to the seven species studied; consensus sequences were analyzed by homology and deposited in the GenBank |
| format | Online |
| id | azm-article-1008 |
| institution | Acta Zoológica Mexicana |
| language | spa |
| publishDate | 2017 |
| publisher | Instituto de Ecología, A.C. |
| record_format | ojs |
| spelling | azm-article-10082022-11-11T23:07:24Z Standardization and validation of the nested pcr test for the diagnosis of the genus xyleborus species (coleoptera: curculionidae: scolytinae) Estandarización y validación de la prueba de pcr anidada para el diagnóstico de especies del género xyleborus (coleoptera: curculionidae: scolytinae) Sosa-Castillo, María Elena Lara Reyna, Joel Ortega Arenas, Laura Delia Judith Hernández, Alfonsina Ambrosial beetles molecular identification diagnosis CO1 gene Escarabajos ambrosiales identificación molecular diagnóstico gen CO1 . In this study it was standardized and validated the nested PCR test for rapid, sensitive and reliable detection of species of the genus Xyleborus using external primers CI-J-2183 and TL2-N-3014 and internal primers J2210 and N2739 that amplify a band of 500 bp in the region of the gene mitochondrial Cytochrome Oxidase subunit 1(CO1). Also, DNA extraction from 26 specimens of Xyleborus was realized with kit Qiagen DNeasy ® mericom Food (DMF), not previously reported its use for their application in insects, which resulted in enough DNA and high-quality for amplification by PCR reactions. The method allowed to process a single insect by extraction, and obtain genetic material from specimens preserved in alcohol of up to eight years old. The detection limit was defined up to a concentration of 780 pg/uL. The PCR was optimized in a final volume of 15 µL without compromising quality of amplification. The standardized test allowed quality DNA, which ensured high reproducibility and sensitivity in the detection of species of Xyleborus and partial sequencing of the CO1 gene to the seven species studied; consensus sequences were analyzed by homology and deposited in the GenBank En este estudio se estandarizó y validó la técnica de PCR anidada para la detección rápida, sensible y confiable de especies del género Xyleborus mediante el uso de los primers externos CI-J-2183 y TL2-N-3014 e internos J2210 y N2739, que amplifican una banda de 500 pb de la región del gen mitocondrial Citocromo Oxidasa subunidad 1(CO1). Asimismo, se realizó la extracción de ADN de 26 ejemplares de Xyleborus con el kit Qiagen DNeasy® mericom Food (DMF), no reportado previamente su uso para su aplicación en insectos, que resultó en ADN suficiente y de alta calidad para reacciones de amplificación por PCR. El método permitió procesar un solo insecto por extracción, y obtener material genético de muestras conservadas en alcohol de hasta ocho años de antigüedad. El límite de detección se definió hasta una concentración de 780 pg/ul. Se optimizóla PCR en un volumen final de 15 uL sin comprometer calidad de la amplificación. La técnica estandarizada permitió la obtención de ADN de calidad, lo que aseguró alta reproducibilidad y sensibilidad en la detección de especies de Xyleborus y la secuenciación parcial del gen CO1 para las siete especies estudiadas; las secuencias consenso fueron analizadas por homología y depositadas en el GenBank. Instituto de Ecología, A.C. 2017-05-23 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion Original articles Artículos originales application/pdf text/html https://azm.ojs.inecol.mx/index.php/azm/article/view/1008 10.21829/azm.2017.3311008 ACTA ZOOLÓGICA MEXICANA (N.S.); Vol. 33 No. 1 (2017); 18-26 ACTA ZOOLÓGICA MEXICANA (N.S.); Vol. 33 Núm. 1 (2017); 18-26 2448-8445 0065-1737 spa https://azm.ojs.inecol.mx/index.php/azm/article/view/1008/1170 https://azm.ojs.inecol.mx/index.php/azm/article/view/1008/1194 Derechos de autor 2017 ACTA ZOOLÓGICA MEXICANA (N.S.) http://creativecommons.org/licenses/by-nc-sa/4.0 |
| spellingShingle | Sosa-Castillo, María Elena Lara Reyna, Joel Ortega Arenas, Laura Delia Judith Hernández, Alfonsina Standardization and validation of the nested pcr test for the diagnosis of the genus xyleborus species (coleoptera: curculionidae: scolytinae) |
| title | Standardization and validation of the nested pcr test for the diagnosis of the genus xyleborus species (coleoptera: curculionidae: scolytinae) |
| title_full | Standardization and validation of the nested pcr test for the diagnosis of the genus xyleborus species (coleoptera: curculionidae: scolytinae) |
| title_fullStr | Standardization and validation of the nested pcr test for the diagnosis of the genus xyleborus species (coleoptera: curculionidae: scolytinae) |
| title_full_unstemmed | Standardization and validation of the nested pcr test for the diagnosis of the genus xyleborus species (coleoptera: curculionidae: scolytinae) |
| title_short | Standardization and validation of the nested pcr test for the diagnosis of the genus xyleborus species (coleoptera: curculionidae: scolytinae) |
| title_sort | standardization and validation of the nested pcr test for the diagnosis of the genus xyleborus species (coleoptera: curculionidae: scolytinae) |
| url | https://azm.ojs.inecol.mx/index.php/azm/article/view/1008 |
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