[Cloning of kDNA minicircles in different species of Leishmania and its use as probes for diagnosis].
The present study describes the cloning procedure for fragments of kinetoplast DNA minicircles from different Leishmania species and its use for detecting the presence of these parasites. Our methodology was as follow: the DNA of the kinetoplast from Leishmania mexicana amazonensis and Leishmania braziliensis panamensis was extracted, purified and digested with the enzyme Dra I. These fragments were cloned in the site for Hinc II in the plasmid pKS. E. coli was the bacterial strain used for transforming and amplifying the cloned fragments; the selection was carried out in LB medium supplemented with ampicillin. With the clones suspected to be positives we run a Southern blot and total kDNA, from each Leishmania species, was used as hybridization probe. Finally, the cloned purified fragments were tested as diagnostic probes against kDNA from eleven different species of Leishmania and one of Trypanosoma cruzi parasites. After cloning, transforming, amplifying and selecting, we obtained two probes of fragments of kDNA minicircles: one from L. m. amazonensis and the other from L. b. panamensis. Both probes showed high sensitivity for diagnosing cutaneous Leishmania complexes (Mexicana or Braziliensis); however, we observed a low grade crossreaction between some species belonging to the same complex. It is necessary to continue studies in order to obtain subfragments of these probes with a higher grade of specificity at the level of species and subspecies.
Pascale
J M
JM
Centro de Investigación y Diagnóstico de Enfermedades Parasitarias (CIDEP), Facultad de Medicina, Universidad de Panamá.
Sousa
O E
OE
López
U
U
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D015342:DNA Probes; D016054:DNA, Protozoan; D006801:Humans; D007891:Leishmania / Q000235:genetics*; D007896:Leishmaniasis / Q000175:diagnosis*; D009693:Nucleic Acid Hybridization
D015342:DNA Probes; D016054:DNA, Protozoan
Clonaje de minicírculos de kADN en diferentes especies de Leishmania y su uso como sondas para diagnóstico.