Isolation and characterization of a repetitive DNA sequence from Leishmania infantum: development of a visceral leishmaniasis polymerase chain reaction

• Published in: The American journal of tropical medicine and hygiene Vol. 49; no. 3; p. 364
• Main Authors: Piarroux, R, Azaiez, R, Lossi, A M, Reynier, P, Muscatelli, F, Gambarelli, F, Fontes, M, Dumon, H, Quilici, M
• Format: Journal Article
• Language: English
• Published: United States 01.09.1993
• Subjects: Animals; Base Sequence; Blotting, Southern; Cloning, Molecular; DNA Probes; DNA, Protozoan - chemistry; DNA, Protozoan - isolation & purification; Dogs; Electrophoresis, Polyacrylamide Gel; Genetic Vectors; Humans; Leishmania donovani - genetics; Leishmaniasis, Visceral - diagnosis; Leishmaniasis, Visceral - parasitology; Molecular Sequence Data; Oligonucleotides - chemistry; Polymerase Chain Reaction; Repetitive Sequences, Nucleic Acid; Restriction Mapping; Sensitivity and Specificity; Species Specificity
• ISSN: 0002-9637
• DOI: 10.4269/ajtmh.1993.49.364

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).