Parasitology

Research Article

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

A. M. MONTALVOa1, J. FRAGAa1, L. MONZOTEa1, I. MONTANOa1, S. DE DONCKERa2, J. C. DUJARDINa2a3 and G. VAN DER AUWERAa2 c1

a1 Instituto de Medicina Tropical Pedro Kourí, Departamento de Parasitología, La Habana, Cuba

a2 Institute of Tropical Medicine Antwerp, Antwerp, Belgium

a3 Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium

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.

(Received August 31 2009)

(Revised November 06 2009)

(Revised January 04 2010)

(Accepted January 04 2010)

(Online publication May 05 2010)

Correspondence:

c1 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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