Evaluation of different storage times and preservation methods on phlebotomine sand fly DNA concentration and purity

Abstract

Background: Different methods have been used to preserve phlebotomine sand flies for research purposes, including for taxonomic studies and detection of Leishmania spp. Here, we evaluated the effect of various preservation methods at different storage times on phlebotomine sand fly DNA concentration and purity. Methods: Field-collected phlebotomine sand flies were individually stored in 70% ethanol (G1) and 95% ethanol (G2) at room temperature, 70% ethanol (G3) and 95% ethanol (G4) at 8 °C or frozen dry (i.e. no preservation solution) at - 20 °C (G5). DNA concentration and purity were assessed at various storage times (T1, ≤ 12 h; T2, 3 months; T3, 6 months; T4, 9 months; and T5, 12 months). Fragments of the cytochrome c oxidase subunit 1 (cox1) and cacophony (CAC) genes of phlebotomine sand flies were also amplified. Results: Mean DNA concentration (P = 0.178) and 260/280 purity ratios (P = 0.584) did not vary significantly among various preservation methods and storage times. Within each group, DNA concentration varied in G1 (Kruskal-Wallis H-test, P = 0.009) for T3 vs T4 (Dunn's post-hoc, P < 0.05), and in G2 (Kruskal-Wallis H-test, P = 0.004) for T1 vs T2 and T1 vs T4 (Dunn's post-hoc, P < 0.05). For 260/280 purity ratios, the only statistically significant difference was found for G5 (Kruskal-Wallis H-test, P = 0.020) between T1 vs T4 (Dunn's post-hoc test, P < 0.05). The cox1 and CAC genes were successfully amplified, regardless of the preservation method and storage time; except in one sample from G2 at T1, for which the CAC gene failed to amplify. Conclusions: The preservation methods and storage times herein evaluated did not affect the concentration and purity of DNA samples obtained from field-collected phlebotomine sand flies, for up to 12 months. Furthermore, these preservation methods did not interfere with PCR amplification of CAC and cox1 genes, being suitable for molecular analyses under the conditions studied herein.[Figure not available: see fulltext.]