[PCR-RFLP and RAPD for typing neotropical Leishmania].
BACKGROUND
The analysis of the PCR-restriction fragment length polymorphism and random amplified polymorphic DNA have been useful tools for Leishmania identification.


OBJECTIVE
Molecular procedures were demonstrated for identification and typing of reference strains of New World Leishmania and their applicability was validated for clinical samples.


METHODS
DNA was extracted from 16 reference strains of Latin American Leishmania as well as from clinical samples of leishmaniasis patients. A sequence coding for cysteine proteinase B was amplified by PCR and subjected to restriction fragment length polymorphism analysis. The enzyme used was Taq1. For eight of the reference strains, the random amplified polymorphic desoxyribonucleic acid technique (RAPD) was applied. Band patterns for Leishmania species differentiation were established each each method. The sample size of the clinical sample was of 5.


RESULTS
PCR products of the cysteine proteinase B gene were obtained for L. braziliensis, L. peruviana, L. panamensis and L. guyanensis. For the other species, L. mexicana, L. amazonensis, L. garnhami, L. lainsoni, L. chagasi, L. naiffi, no amplification occurred. The patterns of restriction fragments revealed band patterns in common for L. peruviana, L. guyanensis and L. panamensis, whereas L. braziliensis had a distinctive pattern. When human samples were examined, amplification occurred for all cases, and the profiles corresponded to the common profile of L. peruviana, L. guyanensis and L. panamensis. The RAPD technique demonstrated reproducible and distinctive patterns for each of the 8 reference strains, L. mexicana, L. amazonensis, L. garnhami, L. lainsoni, L. chagasi, L. naiffi, making possible to differentiate all them. The advantages and limitations of each procedure are discussed.


CONCLUSIONS
The combination of RFP and RAPD methodologies provide useful tools to identify medical important species of Leishmania by recognizing DNA sequences characteristic of each species.
Montalvo
Ana Margarita
AM
Departamento de Parasitología, Instituto Pedro Kourí, La Habana, Cuba. amontalvo@ipk.sld.cu
Monzote
Lianet
L
Fraga
Jorge
J
Montano
Ivón
I
Muskus
Carlos
C
Marín
Marcel
M
de Doncker
Simonne
S
Vélez
Iván Darío
ID
Dujardin
Jean Claude
JC
D000818:Animals; D006801:Humans; D007891:Leishmania / Q000145:classification / Q000235:genetics; D007896:Leishmaniasis / Q000209:etiology / Q000503:physiopathology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D019105:Random Amplified Polymorphic DNA Technique; D014329:Tropical Climate