DNA extraction and PCR amplification

DNA extraction was performed using a Gentra Puregene QIAGEN (Germantown, MD, EUD) extraction kit following the manufacturer’s specifications.

PCR was conducted according to Degrave et al., [20] using the generic primers A: 5’ (C/G)(C/G)(G/C) CC(C/A) CTA T(T/A)T TAC ACC AAC CCC 3’ and B: 5’ GGG GAG GGG CGT TCT GCG AA 3’ in order to amplify a 120bp fragment of the conserved region within the minicircles of Leishmania kDNA [20]. Reaction mixtures were prepared in a final volume of 25uL containing 2uL of DNA template and 1× buffer solution (1.0mM Tris–HCl; 5.0mM KCl; 1.5mM MgCl2; pH 8.0), 200_M dNTPs, 10 pmol of each primer and 1.25U of Taq DNA polymerase (Invitrogen). The amplification conditions were as follows: 94°C for 4 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 30 s, with a final extension step at 72°C for 10min. Amplification were analysed on 2% polyacrylamide gels. All samples were tested for irbp-PCR as internal control for mammal DNA [21, 22] (S1 Appendix).

Amplified products were visualized in a 2% agarose gel stained with ethidium bromide, with a 100 bp DNA Step Ladder provided as a molecular-weight size-standard, and analyzed using the L-PIX EX (Loccus, Biotechnology, Cotia, SP) photo-documentation system. A panel of reference strains was used as a positive control, in all PCR procedures, which included Leishmania amazonensis (IFLA/BR/67/PH8), Le. braziliensis (MHOM/BR/75/M2903), Le. infantum (MHOM/BR/74/PP75) and Le. guyanensis (MHOM/BR/75/M4147).