Polymerase chain reaction-based identification of New World Leishmania species complexes by specific kDNA probes

Here we define a new approach for the detection and characterisation of Lei shmania complexes by polymerase chain reaction (PCR) and specific hybridisa tion. The first step consists of PCR amplification of kDNA minicircles usin g general kinetoplastid primers, which generate a polymorphic multi-banding pattern for all Leishmania species and other Kinetoplastidae. The second s tep is the identification of the Leishmania species complexes by hybridisat ion of the PCR products with specific kDNA probes. Polymorphic PCR-products from a genetically diverse set of Leishmania species were analysed by elec trophoresis and the banding patterns compared with multi-locus enzyme elect rophoresis (MLEE) data. The banding patterns produced by Leishmania species were very heterogeneous, making kDNA-PCR useful for determining closely re lated strains and for fingerprinting individual strains. The degree of kDNA -PCR and MLEE polymorphism was compared using UPGMA dendrograms. Three comp lex-specific probes were generated from major PCR bands of reference stocks belonging to the Leishmania mexicana, Leishmania donovani and Leishmania b raziliensis complexes, and hybridisation of these probes to membrane-bound PCR products could reliably identify the strain to a complex level. A combi nation of kDNA-PCR fingerprinting and hybridisation with kDNA probes was fo und to be useful for both sensitive detection and direct identification of Leishmania species complexes. (C) 1999 Elsevier Science B.V. All rights res erved.