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).
Laboratoire de Parasitologie, Faculte de Medecine, Marseille, France.
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Piarroux
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Azaiez
A M
AM
Lossi
P
P
Reynier
F
F
Muscatelli
F
F
Gambarelli
M
M
Fontes
H
H
Dumon
M
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Quilici
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