Leishmania infantum species-specific kDNA probes: isolation and evaluation.
The study refers to the isolation of specific DNA probes to the parasite species Leishmania (L) infantum according to different strategies using recombinant minicircles isolated from L. infantum kinetoplast DNAs. A first probe was identified following a classical procedure. One mini-circle selected for strong reactivity to L. infantum total DNA was used to identify specific subfragments to this species among which the 95bp fragment, 3B8HaeIII-2 was selected. For the obtention of the second probe, a strategy based on sequential screenings for specificity and sensitivity was applied. This allowed identification of a set of minicircles showing an increased specificity to L. infantum as compared to other species, and an increased sensitivity of reaction as compared to the other minicircles. Subclonings and screenings allowed a final selection of a 137bp-minicircle fragment: 3E9HaeIII-12. Reactivities of the 2 probes were assessed on a panel of total DNAs and promastigotes from 74 isolates pertaining to 9 species encountered in the Old World. Parasites isolated in Tunisia from different foci, different hosts after different transmission seasons were included. Hybridizations have shown the exquisite specificity of these probes to L. infantum in this country. Probe 3E9HaeIII-12 was found to be the more sensitive where down to 10 ng of total DNA and 10(3) promastigotes could be detected. From this study and as compared to data provided in the literature, the second procedure allowed at least 10-fold increase in sensitivity.
Ben Hamouda
A
A
Laboratoire d'Epidémiologie et d'Ecologie Parasitaire Institut Pasteur de Tunis-BP 74-1002 Tunis Belvédère, Tunisia.
Guizani
I
I
Jedidi
S
S
Dellagi
K
K
Ben Ismaïl
R
R
D000818:Animals; D015342:DNA Probes / Q000235:genetics / Q000302:isolation & purification; D018105:DNA, Kinetoplast / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics* / Q000302:isolation & purification; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D006801:Humans; D006824:Hybridization, Genetic / Q000235:genetics; D015151:Immunoblotting; D018314:Leishmania infantum / Q000235:genetics*; D007898:Leishmaniasis, Visceral / Q000175:diagnosis* / Q000453:epidemiology / Q000469:parasitology / Q000635:transmission; D012621:Seasons; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014416:Tunisia / Q000453:epidemiology
D015342:DNA Probes; D018105:DNA, Kinetoplast; D004274:DNA, Recombinant; D015252:Deoxyribonucleases, Type II Site-Specific; C037130:GCGC-specific type II deoxyribonucleases; C024403:GGCC-specific type II deoxyribonucleases