Isolation and characterization of a repetitive DNA sequence from Leishmania infantum: development of a visceral leishmaniasis polymerase chain reaction.
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).
Piarroux
R
R
Laboratoire de Parasitologie, Faculte de Medecine, Marseille, France.
Azaiez
R
R
Lossi
A M
AM
Reynier
P
P
Muscatelli
F
F
Gambarelli
F
F
Fontes
M
M
Dumon
H
H
Quilici
M
M
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D015342:DNA Probes; D016054:DNA, Protozoan / Q000737:chemistry* / Q000302:isolation & purification; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D007893:Leishmania donovani / Q000235:genetics*; D007898:Leishmaniasis, Visceral / Q000175:diagnosis / Q000469:parasitology*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D012680:Sensitivity and Specificity; D013045:Species Specificity
D015342:DNA Probes; D016054:DNA, Protozoan; D009841:Oligonucleotides