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Open AccessProtocol

Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification

Centro Regional de Estudios Genómicos, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata 1900, Argentina
Departamento de Informática y Tecnología, Universidad Nacional del Noroeste de la Provincia de Buenos Aires, Pergamino, Buenos Aires 2700, Argentina
Laboratorio de Vectores, Secretaría de Calidad de Vida, Municipalidad de Posadas, Posadas, Misiones 3300, Argentina
Instituto Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia 40296-710, Brasil
Instituto Leonidas e Maria Deane, Fundação Oswaldo Cruz, Manaus, Amazônia 69057-070, Brasil
Centro Nacional de Diagnóstico e Investigación en Endemoepidemias, Administración Nacional de Laboratorios e Institutos de Salud, Ministerio de Salud, Buenos Aires 1063, Argentina
Instituto Nacional de Medicina Tropical, Ministerio de Salud de la Nación, Puerto Iguazú, Misiones 3370, Argentina
Laboratory of Medical Entomology, René Rachou Research Institute, Fundação Oswaldo Cruz, Minas Gerais 30190-002, Brazil
Author to whom correspondence should be addressed.
Methods Protoc. 2019, 2(2), 36;
Received: 13 March 2019 / Revised: 2 May 2019 / Accepted: 3 May 2019 / Published: 7 May 2019
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. View Full-Text
Keywords: sand fly; DNA extraction; calcium; PCR; lysis buffer; Lutzomyia sand fly; DNA extraction; calcium; PCR; lysis buffer; Lutzomyia
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Caligiuri, L.G.; Sandoval, A.E.; Miranda, J.C.; Pessoa, F.A.; Santini, M.S.; Salomón, O.D.; Secundino, N.F.C.; McCarthy, C.B. Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification. Methods Protoc. 2019, 2, 36.

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