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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 
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.
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.
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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