Evaluating the specificity of primers employed for PCR-based diagnostics of Leishmaniases using multiple alignment analysis

The purpose of this study was to assess the specificity of distinct primers used in the molecular diagnosis for Leishmania detection by using biological sequence search and alignment tools. Four primer pairs routinely employed in the PCR-based diagnostics for Leishmaniasis detection were evaluated through the software Primer-BLAST, which compares nucleotide sequences against user-selected database to avoid primer pairs cause non-specific amplifications. The LU5A-LB3C primer pair showed good specificity among all primers analyzed, generating alignments exclusively against distinct sequences of Leishmania species and no matches were found with other parasite species. Whereas the primer pairs designated MP3H - MP1L, B1-B2 and 13A-13B demonstrated matches against distinct sequences of Leishmania strains and other species, such as Rhesus monkey (Macaca mulatta) and starlet sea anemone (Nematostella vectensis). These findings emphasize the importance of selecting suitable primers for diagnosis of molecular diseases by conducting previous screenings in order to infer their specificity and identity against target templates within biological sequence annotation database.