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Abstract
BackgroundDifferent 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.MethodsField-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.ResultsMean 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.ConclusionsThe 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.
Highlights
Different methods have been used to preserve phlebotomine sand flies for research purposes, includ‐ ing for taxonomic studies and detection of Leishmania spp
We evaluated the effect of various preservation methods involving the use of ethanol on the concentration and purity of phlebotomine sand fly deox‐ yribonucleic acid (DNA) for up to 12 months
The highest mean DNA concentration was found in G2 and the lowest in G3, with DNA concentrations ranging from 2.7–8.2 ng/μl and 2.3–4.2 ng/μl, respectively, depending on storage time (Fig. 1)
Summary
Different methods have been used to preserve phlebotomine sand flies for research purposes, includ‐ ing for taxonomic studies and detection of Leishmania spp. Phlebotomine sand flies are dipterans of medical and veterinary significance, due to their ability to transmit disease agents of various animal species, including humans [1]. While they transmit viruses and bacteria, they are mostly known as biological vectors of Leishmania spp. parasites, which cause approximately 0.2–0.4 million cases of visceral leishmaniasis and 0.7–1.2 million cases of cutaneous leishmaniasis, in 98 countries every year [2]. Phlebotomine sand flies inhabit various types of environments, including caves, forests, crop plantations and human houses [3] Some of these environments are difficult to access and far from research centres, making their transportation and preservation a critical step in research projects focused on the biology, taxonomy, genetics, and vector role of these insects
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