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Background  Leishmaniasis is a neglected disease caused by many  Leishmania  species, belonging to subgenera  Leishmania  ( Leishmania ) and  Leishmania  ( Viannia ) .  Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of  Leishmania  species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as the target sequence. These assays had potential cross-species amplification, due to sequence similarity between  Leishmania  species. Previous works demonstrated discrimination between  L.  ( Leishmania ) and  L.  ( Viannia ) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high-resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish  L.  ( L .)  infantum  from  L.  ( L .)  amazonensis , which can coexist in the same geographical area.    Methods  DNA from 18 strains/isolates of  L.  ( L .)  infantum ,  L.  ( L .)  amazonensis ,  L.  ( V. )  braziliensis ,  L.  ( V. )  panamensis ,  L.  ( V. )  guyanensis , and 62 clinical samples from  L.  ( L .)  infantum -infected dogs were amplified by a previously developed qPCR (qPCR-ML) and subjected to HRM analysis; selected PCR products were sequenced using an ABI PRISM 310 Genetic Analyzer. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in  L.  ( L .)  amazonensis  was designed.    Results  The qPCR-ML followed by HRM analysis did not allow discrimination between  L.  ( L .)  amazonensis  and  L.  ( L .)  infantum  in 53.4% of cases. Hence, the novel SYBR green-based qPCR (qPCR-ama) has been tested. This assay achieved a detection limit of 0.1 pg of parasite DNA in samples spiked with host DNA and did not show cross amplification with  Trypanosoma cruzi  or host DNA. Although the qPCR-ama also amplified  L.  ( L .)  infantum  strains, the C q  values were dramatically increased compared to qPCR-ML. Therefore, the combined analysis of C q  values from qPCR-ML and qPCR-ama allowed to distinguish  L.  ( L .)  infantum  and  L.  ( L .)  amazonensis  in 100% of tested samples.    Conclusions  A new and affordable SYBR-green qPCR-based approach to distinguish between  L.  ( L .)  infantum  and  L.  ( L .)  amazonensis  was developed exploiting the major abundance of a minicircle sequence rather than targeting a hypothetical species-specific sequence. The fast and accurate discrimination between these species can be useful to provide adequate prognosis and treatment.    Electronic supplementary material  The online version of this article (doi:10.1186/s13071-017-2181-x) contains supplementary material, which is available to authorized users.

The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis