Nuclear DNA sequence specific to Leishmania (Viannia) subgenus:: a molecular marker for species identification

As shown by RFLP analysis, there is a high variability in the beta -tubulin gene region of Leishmania sp. Such variability has been used in the identification of these parasites, establishing differences between subgenera of New World Leishmania. We have found a region of 500 bp (beta 500) upstream of the coding region of the beta -tubulin gene that is present in all strains tested belonging to the L. (Viannia) subgenus. This region apparently is a repetitive sequence and we have shown that it is specific to the Leishmania (Viannia) subgenus. This sequence has no homology with the genomic DNA isolated from either the species belonging to the L. (Leishmania) subgenus or other Kinetoplastida, such as Trypanosoma cruzi, T. brucei, Leptomonas samueli, or Crithidia fasciculata. The beta 500 sequence showed sufficient variation to be used as a molecular marker in the identification of parasites. We established inter- and intrasubgenus differentiation and were able to discriminate at the species lever in the Vianna subgenus. A PCR assay confirmed the specificity of the beta 500 sequence.