Standardization and validation of the nested pcr test for the diagnosis of the genus xyleborus species (coleoptera: curculionidae: scolytinae)
DOI:
https://doi.org/10.21829/azm.2017.3311008Keywords:
Ambrosial beetles, molecular identification, diagnosis, CO1 geneAbstract
. 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 GenBankDownloads
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Copyright (c) 2017 María Elena Sosa-Castillo, Joel Lara Reyna, Laura Delia Ortega Arenas, Alfonsina Judith Hernández
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