Table 1.
DNA sequence and genomic region of molecular targets employed in the multiplex PCR system.
Table 2.
Details of the samples employed to validate the multiplex PCR system: Mammal order, geographic origin and tissue from wild mammalian hosts previously diagnosed with Leishmania spp. infection using a singleplex reaction.
Fig 1.
gapdh alignment among mammal and Leishmania species and phylogenetic tree of the gapdh sequences.
(A) Multiple alignment of the target gapdh segment (212 bp) of different mammals and Leishmania species using ClustalW in CLC-Main Workbench software (6.9.1). All sequences were accessed from the NCBI and EMBL-EBI databases using UniProt and InterPro resources. The alignment showed 122 (57%) nucleotides that were conserved in different species, whereas 167 (79%) were conserved in mammal sequences (variable height in the bar plot). (B) A phylogenetic tree of the target gapdh sequence (212 bp) of different mammal and Leishmania species based on the neighbor joining method showed two divergent branches: mammals and Leishmania clusters. Arrows—localization and orientation of each primer in the alignment; bar plot—nucleotide conservation; consensus—sequence generated through the most conserved nucleotides between all aligned sequences; tree parameters: neighbor joining tree with 1,000 replicates and constructed based on the Kimura 80 distance measure using CLC-Main Workbench software (6.9.1).
Fig 2.
Silver-stained electrophoresis polyacrylamide gel obtained from the amplification of gapdh and kDNA with the multiplex PCR system.
(A) PCR products from the multiplex PCR system using Taq DNA polymerase enzymes (ABM®) in the presence of 5% DMSO. (B) PCR products from the multiplex PCR system using the FideliTaq PCR Master Mix (Affymetrix, USB). In both figures: 1. molecular-weight marker (50-bp DNA ladder); 2. infected rodent; 3. uninfected rodent; 4. human DNA; and 5. negative PCR control.
Fig 3.
Silver-stained electrophoresis polyacrylamide gel obtained from the sensitivity test for detecting Leishmania sp. DNA with a constant concentration of human DNA (100 ng).
1: molecular-weight marker (50-bp DNA ladder); 2. empty; 3–8. mixture of human DNA and 100 (3), 50 (4), 10 (5), 5 (6), 1 (7) and 0.1 (8) ng of L. tropica; 9. uninfected rodent; 10. Leishmania tropica DNA; 11. Leishmania tarentolae DNA; and 12. Negative control for the PCR.
Table 3.
Amplification of the molecular targets used in the multiplex PCR system using samples from wild mammalian hosts.