| Autor: |
Caligiuri LG; Centro Regional de Estudios Genómicos, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata 1900, Argentina. lgcaligiuri@gmail.com.; Departamento de Informática y Tecnología, Universidad Nacional del Noroeste de la Provincia de Buenos Aires, Pergamino, Buenos Aires 2700, Argentina. lgcaligiuri@gmail.com., Sandoval AE; Laboratorio de Vectores, Secretaría de Calidad de Vida, Municipalidad de Posadas, Posadas, Misiones 3300, Argentina. sandoval@inti.gob.ar., Miranda JC; Instituto Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia 40296-710, Brasil. jmiranda@bahia.fiocruz.br., Pessoa FA; Instituto Leonidas e Maria Deane, Fundação Oswaldo Cruz, Manaus, Amazônia 69057-070, Brasil. favpessoa@gmail.com., Santini MS; Centro Nacional de Diagnóstico e Investigación en Endemoepidemias, Administración Nacional de Laboratorios e Institutos de Salud, Ministerio de Salud, Buenos Aires 1063, Argentina. mariasoledadsantini@gmail.com., Salomón OD; Instituto Nacional de Medicina Tropical, Ministerio de Salud de la Nación, Puerto Iguazú, Misiones 3370, Argentina. odanielsalomon@gmail.com., Secundino NFC; Laboratory of Medical Entomology, René Rachou Research Institute, Fundação Oswaldo Cruz, Minas Gerais 30190-002, Brazil. secundinon@gmail.com., McCarthy CB; Centro Regional de Estudios Genómicos, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata 1900, Argentina. mccarthychristina@gmail.com.; Departamento de Informática y Tecnología, Universidad Nacional del Noroeste de la Provincia de Buenos Aires, Pergamino, Buenos Aires 2700, Argentina. mccarthychristina@gmail.com. |
| Abstrakt: |
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 Ca 2+ (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 Ca 2+ has been reported for the extraction of DNA from sand flies. |
