Development of species-specific multiplex PCR for Leishmania identification

Leishmaniasis is a diverse group of clinical diseases caused by protozoan parasites of the Leishmania genus. Species-specific identification of Leishmania spp. is challenging due to the high number of different pathogenic species that sometimes co-circulate in the same foci, hampering efforts to effectively control the disease. Multiplex PCR is an attractive alternative for rapid differentiation of Leishmania species with high sensitivity and specificity. We aimed to generate a panel of primers optimized for a multiplex PCR assay capable of identifying different Leishmania species in a single reaction. Species-specific primers were designed based on genomic data using the TipMT tooL. Potential non-specific amplifications of other trypanosomatids as well as human, dog, and sandfly hosts were first evaluated in silico using the Primer-Blast tooL. Species-specific primers for Leishmania amazonensis, Leishmania braziliensis, Leishmania donovani, Leishmania infantum, Leishmania mexicana and for the Leishmania guyanensis complex were tested in vitro. The primers have a limit of detection ranging from 1 to 0.01 ng of parasite gDNA using the same annealing temperature of 66 °C. The primers were specific for their targets when tested against 13 species of Leishmania, six trypanosomatids, and Babesia sp., and to detect the target species in a prepared pool with gDNA of six pathogenic Leishmania species. The designed primers were optimized for multiplex PCR, enabling species-specific identification of all five Leishmania species and one species complex. This new primer set could allow for efficient, fast, and reliable identification of Leishmania parasites.