Preprint Article Version 1 This version is not peer-reviewed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)

How to cite: Caligiuri, L.G..; Sandoval, A.E...; Miranda, J.C..; Pessoa, F.A..; Santini, M.S..; Salomón, O.D..; Secundino, N.F.C...; McCarthy, C. Optimisation of DNA Extraction from Individual Sand Flies for PCR Amplification. Preprints 2018, 2018120189 (doi: 10.20944/preprints201812.0189.v1). Caligiuri, L.G..; Sandoval, A.E...; Miranda, J.C..; Pessoa, F.A..; Santini, M.S..; Salomón, O.D..; Secundino, N.F.C...; McCarthy, C. Optimisation of DNA Extraction from Individual Sand Flies for PCR Amplification. Preprints 2018, 2018120189 (doi: 10.20944/preprints201812.0189.v1).

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.

Subject Areas

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

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