CONICET | Buscador de Institutos y Recursos Humanos

Biopsy is widely used for the diagnosis of cutaneous leishmaniasis (CL) to obtain specimens for direct diagnoses (smear and culture). Molecular diagnosis supplementconventional diagnosis, although it is not well suited for adoption in laboratories with limited resources were these parasitosis are endemic. Isothermal DNA amplification methods have the advantage of not requiring expensive equipment. The aim of this work was to utilize, a previously reported LAMP assay, to detect CL calorimetrically (Mikita et al., 2014, Rivero et al., 2017). LAMP reactions were performed using pan-Leishmania primers based on the 18S-rDNA sequences. Briefly, DNA samples (5 μL) were subjected to amplification in reaction mixtures containing 40 pmol FIP and BIP primers, 20 pmol LF and LB primers, 5 pmol F3 and B3 primers, 1 μL (8 units) Bst DNA polymerase (New England Biolabs), the reaction buffer (20 mM Tris?HCl, 10 mM KCl, 8 mM MgSO4, 10 mM (NH4)2SO4, 0.1% Tween-20), and 1.4 mM of each dNTP. The LAMP assay was set up testing different concentrations of betaine (0.5, 0.8, 1, or 1.6 M) and temperatures (55, 57, 60, 63 ºC), using a heat block (Labnet®) for the amplification cycle. Two approaches were used to confirm the amplification by using electrophoresis in 1.5 % agarose and by visual inspection after the addition of the fluorescent dye SYBR® Green (Invitrogen, S7563). The detection limit of this procedure was 1.0 × 102 parasites/mL. This assay was tested in 10 samples of patient infected to CL. LAMP was unable to detect genomic DNA from Toxoplasma gondii, Treponema pallidum or Human herpesvirus 5 (HHV-5). The advantages of this novel tool include the speed with which the assays can be completed, the no-need of trained personnel, and the fact that it can be performed without complex and expensive laboratory equipment.