Isolation and Characterization of a Repetitive DNA Sequence from Leishmania infantum: Development of a Visceral Leishmaniasis Polymerase Chain Reaction

R. PiarrouxLaboratoire de Parasitologie, Faculte de Medecine, INSERM U242, Faculte de Medicine, Marseille, France

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R. AzaiezLaboratoire de Parasitologie, Faculte de Medecine, INSERM U242, Faculte de Medicine, Marseille, France

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A. M. LossiLaboratoire de Parasitologie, Faculte de Medecine, INSERM U242, Faculte de Medicine, Marseille, France

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P. ReynierLaboratoire de Parasitologie, Faculte de Medecine, INSERM U242, Faculte de Medicine, Marseille, France

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F. MuscatelliLaboratoire de Parasitologie, Faculte de Medecine, INSERM U242, Faculte de Medicine, Marseille, France

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F. GambarelliLaboratoire de Parasitologie, Faculte de Medecine, INSERM U242, Faculte de Medicine, Marseille, France

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M. FontesLaboratoire de Parasitologie, Faculte de Medecine, INSERM U242, Faculte de Medicine, Marseille, France

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H. DumonLaboratoire de Parasitologie, Faculte de Medecine, INSERM U242, Faculte de Medicine, Marseille, France

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M. QuiliciLaboratoire de Parasitologie, Faculte de Medecine, INSERM U242, Faculte de Medicine, Marseille, France

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To construct a DNA probe specific for protozoa that cause visceral leishmaniasis, we cloned Pst I fragments of Leishmania infantum genomic DNA into a Bluescript II SK® vector. A clone of 4.3 kb that contained a highly repetitive sequence was isolated and cut with three restriction enzymes: Hae III, Rsa I, and Sau 3A. After a new molecular cloning step, we isolated and sequenced a 140-basepair (bp) fragment. Two oligonucleotides were synthesized to be used as primers for a polymerase chain reaction. Using this probe, we detected an amount of DNA equivalent to one promastigote of L. infantum. This probe showed a high specificity; all protozoa tested that cause visceral leishmaniasis and L. major (one of the causative agents of Old World cutaneous leishmaniasis) showed a 100-bp amplified sequence, whereas other Leishmania strains showed a signal of a different size or else no signal. Moreover, no amplified sequence was obtained with other pathogenic parasites tested (Trypanosoma brucei, T. cruzi, Plasmodium falciparum, Pneumocystis carinii, and Toxoplasma gondii).

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