Generation of Specific Dna Probes for Ethiopian Isolates of Leishmania Donovan
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Date
1994-05
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Addis Ababa University
Abstract
In this study the generation of kinetoplast DNA (kDNA) probes specific for Ethiopian
Leishmania donovani are described. kDNA was isolated from cultured L. donovani
promastigotesand digested with restriction endonuclease. The restriction fragments were
cloned into pUCI8 plasmid vector and propagated in Escherichia coli JMI09 competent
strains. Recombinant clones were identified by selecting for plasmid born ampicillin
resistance followed by colony hybridization screening using radiolabelled L. donovani total
kDNA probe. The generated kDNA library was further analyzed by differential
hybridizations to total kDNA probes from L. aefhiopica and L. major. Further screening
was conducted by hybridizing radiolabelled plasmid DNA from the L. donovani kDNA
sequence bearing clones with dot blots of L. donovani, L. major, L. aefhiopica and L.
fropica promoastigotes. Six clones that hybridize only to the parent L. donovani were
selected and analyzed by the same procedure using several L. donovani and other isolates.
Five clones which hybridize with all Ethiopian L. donovani isolates but not to any of the
cutaneous parasites tested, were identified. These L. donovani specific cloned minicircle
kDNA sequences showed various specificities and sensitivities within the L. donovani
complex. One of these sequences -Ld 14, specifically detected Ethiopian and other East
African visceral Leishmania parasites. A polymerase chain reaction (PCR) assay was
designed for the sensitive detection of visceral Leishmania. The kDNA probes and PCR
described in this study may be useful in the detection of L. donovani parasites in Ethiopia.
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Biology