title: Polymerase chain reaction-based identification of New World Leishmania species complexes by specific kDNA probes.
abstract: Here we define a new approach for the detection and characterisation of Leishmania complexes by polymerase chain reaction (PCR) and specific hybridisation. The first step consists of PCR amplification of kDNA minicircles using general kinetoplastid primers, which generate a polymorphic multi-banding pattern for all Leishmania species and other Kinetoplastidae. The second step is the identification of the Leishmania species complexes by hybridisation of the PCR products with specific kDNA probes. Polymorphic PCR-products from a genetically diverse set of Leishmania species were analysed by electrophoresis and the banding patterns compared with multi-locus enzyme electrophoresis (MLEE) data. The banding patterns produced by Leishmania species were very heterogeneous, making kDNA-PCR useful for determining closely related strains and for fingerprinting individual strains. The degree of kDNA-PCR and MLEE polymorphism was compared using UPGMA dendrograms. Three complex-specific probes were generated from major PCR bands of reference stocks belonging to the Leishmania mexicana, Leishmania donovani and Leishmania braziliensis complexes, and hybridisation of these probes to membrane-bound PCR products could reliably identify the strain to a complex level. A combination of kDNA-PCR fingerprinting and hybridisation with kDNA probes was found to be useful for both sensitive detection and direct identification of Leishmania species complexes.
authors.affiliation: UMR CNRS/ORSTOM no. 9926: Génétique Moléculaire des Parasites et des Vecteurs, ORSTOM, Montpellier, France.
authors.forename: S F
authors.initials: SF
authors.lastname: Brenière
authors.forename: J
authors.initials: J
authors.lastname: Telleria
authors.forename: M F
authors.initials: MF
authors.lastname: Bosseno
authors.forename: R
authors.initials: R
authors.lastname: Buitrago
authors.forename: B
authors.initials: B
authors.lastname: Bastrenta
authors.forename: G
authors.initials: G
authors.lastname: Cuny
authors.forename: A L
authors.initials: AL
authors.lastname: Banuls
authors.forename: S
authors.initials: S
authors.lastname: Brewster
authors.forename: D C
authors.initials: DC
authors.lastname: Barker
mesh_terms: D000818:Animals; D001483:Base Sequence; D015342:DNA Probes; D018105:DNA, Kinetoplast / Q000235:genetics*; D004586:Electrophoresis / Q000379:methods; D004798:Enzymes / Q000032:analysis; D007891:Leishmania / Q000145:classification* / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D015342:DNA Probes; D018105:DNA, Kinetoplast; D004798:Enzymes