Recombinant kinetoplast DNA (kDNA) probe for identifying Leishmania tropica

Abstract

The paper reports on the construction of a kDNA library with DNA isolated from the WHO reference strain of Leishmania tropica IC-305 and subsequent identification and propagation of recombinant plasmids containing L. tropica kDNA sequences. It also shows that the cloned kDNA sequences can be used as genetic markers in restriction endonuclease, Southern blot transfer, and dot blot hybridisation analysis, to identify L. tropica parasites. When the pL 305-I kDNA probe was used in hybridisation experiments with DNAs from various Leishmania reference strains, species and isolates from different host species and from different geographical locations, hybridisation was detected only with L. tropica, thereby suggesting that the insert in recombinant plasmid 305-I was species-specific. The probe is sensitive to the level of 10 3 parasites in dot blot hybridisation. Additionally, orthogonal field alternation gel electrophoresis (OFAGE) and transverse alternating field electrophoresis (TAFE) were used to characterise Leishmania reference strains and Leishmania species. The molecular karyotypes resolved by these techniques showed significant differences in the profiles of chromosomal sized-DNA molecules among species of Leishmania. The DNA karyotypes of the two reference strains of L. tropica (IC-305 and NLB-067), while similar, were nevertheless distinct.