Identification of Pathogenic Leishmania Promastigotes by DNA: DNA Hybridization with Kinetoplast DNA Cloned into E. Coli Plasmids

View More View Less
  • Codon, 430 Valley Drive, Brisbane, California 94005
  • | Department of Immunology, Walter Reed Army Institute of Research, Washington, D.C. 20307

We report the characterization of Leishmania (L. infantum, L. donovani, and L. major) kinetoplast DNA (kDNA) by the use of restriction endonuclease digestion patterns and Southern hybridizations. Overall, the sizes and fragment patterns of MspI restriction endonuclease-produced DNA fragments vary from species to species. However, kDNA isolates from different species and strains cross-reacted to a great extent in Southern hybridization experiments. Only kDNA isolated from L. infantum and L. major had little homology during hybridization reactions. To prepare DNA probes that would differentiate between species of Leishmania, minicircle kDNA was digested with restriction enzymes and ligated to an E. coli plasmid. Several plasmids were isolated that specifically detect in hybridization experiments as few as 5 × 103 L. donovani or L. infantum promastigotes lysed on nitrocellulose filters.