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Open AccessProtocol

Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification

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Centro Regional de Estudios Genómicos, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata 1900, Argentina
2
Departamento de Informática y Tecnología, Universidad Nacional del Noroeste de la Provincia de Buenos Aires, Pergamino, Buenos Aires 2700, Argentina
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Laboratorio de Vectores, Secretaría de Calidad de Vida, Municipalidad de Posadas, Posadas, Misiones 3300, Argentina
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Instituto Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia 40296-710, Brasil
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Instituto Leonidas e Maria Deane, Fundação Oswaldo Cruz, Manaus, Amazônia 69057-070, Brasil
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Centro Nacional de Diagnóstico e Investigación en Endemoepidemias, Administración Nacional de Laboratorios e Institutos de Salud, Ministerio de Salud, Buenos Aires 1063, Argentina
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Instituto Nacional de Medicina Tropical, Ministerio de Salud de la Nación, Puerto Iguazú, Misiones 3370, Argentina
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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; https://doi.org/10.3390/mps2020036
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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