Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Optimisation of DNA Extraction from Individual Sand Flies for PCR Amplification

Version 1 : Received: 14 December 2018 / Approved: 17 December 2018 / Online: 17 December 2018 (10:41:28 CET)

A peer-reviewed article of this Preprint also exists.

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. 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.

DOI: 10.3390/mps2020036

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 analysing parasite infection in Lutzomyia spp. by PCR [1] and, for this reason, we evaluated various modifications on a previously published protocol ([2] and Acardi personal communication). The most significant variation was the use of a different lysis buffer [3] to which added Ca2+ (buffer TESCa), because this ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site [4]. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene [5,6]. 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.

Keywords

Sand fly; DNA extraction; Calcium; PCR, lysis buffer, Lutzomyia

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