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Heat-shock protein 70 PCR-RFLP: a universal simple tool for Leishmania species discrimination in the New and Old World

Published online by Cambridge University Press:  05 May 2010

A. M. MONTALVO ,
J. FRAGA ,
L. MONZOTE ,
I. MONTANO ,
S. DE DONCKER ,
J. C. DUJARDIN  and
G. VAN DER AUWERA
A. M. MONTALVO
Affiliation:
Instituto de Medicina Tropical Pedro Kourí, Departamento de Parasitología, La Habana, Cuba
J. FRAGA
Affiliation:
Instituto de Medicina Tropical Pedro Kourí, Departamento de Parasitología, La Habana, Cuba
L. MONZOTE
Affiliation:
Instituto de Medicina Tropical Pedro Kourí, Departamento de Parasitología, La Habana, Cuba
I. MONTANO
Affiliation:
Instituto de Medicina Tropical Pedro Kourí, Departamento de Parasitología, La Habana, Cuba
S. DE DONCKER
Affiliation:
Institute of Tropical Medicine Antwerp, Antwerp, Belgium
J. C. DUJARDIN
Affiliation:
Institute of Tropical Medicine Antwerp, Antwerp, Belgium Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium
G. VAN DER AUWERA*
Affiliation:
Institute of Tropical Medicine Antwerp, Antwerp, Belgium
*
*Corresponding author: Department of Parasitology, Institute of Tropical Medicine Antwerp, Nationalestraat 155, 2000 Antwerp, Belgium. Tel: +32 3 2476586. Fax: +32 3 2476359. E-mail: gvdauwera@itg.be
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Summary

Introduction. Species typing in leishmaniasis gains importance in diagnostics, epidemiology, and clinical studies. A restriction fragment length polymorphism (RFLP) assay of PCR amplicons from a partial heat-shock protein 70 gene (hsp70) had been described for the New World, allowing identification of some species. Methods. Based on an initial in silico analysis of 51 hsp70 sequences, most of which we recently determined in the frame of a phylogenetic study, species-specific restriction sites were identified. These were tested by PCR-RFLP on 139 strains from 14 species, thereby documenting both inter- and intra-species variability. Results. Our assay could identify Leishmania infantum, L. donovani, L. tropica, L. aethiopica, L. major, L. lainsoni, L. naiffi, L. braziliensis, L. peruviana, L. guyanensis, and L. panamensis by applying 2 subsequent digests. L. mexicana, L. amazonensis, and L. garnhami did not generate species-specific restriction fragment patterns. Conclusion. Currently no assay is available for global Leishmania species discrimination. We present a universal PCR-RFLP method allowing identification of most medically relevant Old and New World Leishmania species on the basis of a single PCR, obviating the need to perform separate PCRs. The technique is simple to perform and can be implemented in all settings where PCR is available.

Keywords

LeishmanialeishmaniasisHSP70species typingdifferential diagnosisrestriction fragment length polymorphismPCR-RFLP
Type
Research Article
Information
Parasitology , Volume 137 , Issue 8 , July 2010 , pp. 1159 - 1168
Copyright
Copyright © Cambridge University Press 2010

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