Single-step duplex kDNA-PCR for detection of Leishmania donovani complex in human peripheral blood samples

A duplex polymerase chain reaction (PCR) assay was performed using a sense Leishmania genus-specific oligonucleotides to amplify the conserved region of 120 bp of minicircle kDNA and an additional antisense Leishmania donovani oligonucleotide that amplifies a complex-specific fragment of 90 bp. All 12 tested reference isolates of the L. donovani complex yielded a complex-specific amplification product of 90 bp concurrently with a genus-specific 120-bp product. All 17 tested reference isolates pertaining to other complexes presented only the genus-specific 120-bp product. Peripheral blood samples of twenty patients with visceral leishmaniasis positive by the genus-specific PCR presented both fragments with the duplex assay. This duplex PCR can be applied as a 1-step diagnostic tool where discrimination of L. donovani complex species is relevant for clinical and epidemiologic purposes.