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Title: | Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification | Authors: | Caligiuri, Lorena G Sandoval, Adolfo E Miranda, Jose C Pessoa, Felipe A Santini, María Soledad Salomón, Oscar Daniel Secundino, Nagila F C McCarthy, Christina B. |
Keywords: | Psychodidae;ADN;Reacción en Cadena de la Polimerasa;Calcio | Issue Date: | 7-May-2019 | Journal: | Methods and protocols | Abstract: | Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca2+ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies. |
Description: | Fil: Caligiuri, Lorena G. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro Regional de Estudios Genómicos; Argentina. Fil: Sandoval, Adolfo E. Municipalidad de Posadas. Secretaría de Calidad de Vida. Laboratorio de Vectores, Posadas, Misiones; Argentina. Fil: Miranda, Jose C. Fundação Oswaldo Cruz. Instituto Gonçalo Moniz, Salvador, Bahía; Brasil. Fil: Pessoa, Felipe A. Fundação Oswaldo Cruz. Instituto Leonidas e Maria Deane, Manaos, Amazonas; Brasil. Fil: Santini, María Soledad. ANLIS Dr.C.G.Malbrán. Centro Nacional de Diagnóstico e Investigación en Endemo Epidemias; Argentina. Fil: Salomón, Oscar Daniel. Ministerio de Salud de la Nación. Instituto Nacional de Medicina Tropical, Puerto Iguazú, Misiones; Argentina. Fil: Secundino, Nagila F. C. Fundação Oswaldo Cruz. René Rachou Research Institute. Laboratory of Medical Entomology; Minas Gerais; Brasil. Fil: McCarthy, Christina B. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro Regional de Estudios Genómicos; Argentina. |
URI: | http://sgc.anlis.gob.ar/handle/123456789/1366 | ISSN: | 2409-9279 | DOI: | 10.3390/mps2020036 | Rights: | Open Access Creative Commons Attribution 4.0 International License |
Appears in Collections: | Publicaciones CeNDIE |
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MethodsandProtocols_2019_2_2_p36.pdf | 5.15 MB | Adobe PDF | View/Open |
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