Parasitology

Original Articles

An integrated pipeline for the development of novel panels of mapped microsatellite markers for Leishmania donovani complex, Leishmania braziliensis and Leishmania major

M. FAKHARa1, M. H. MOTAZEDIANa1, D. DALYa2, C. D. LOWEa2, S. J. KEMPa2 and H. A. NOYESa2 c1

a1 Department of Medical Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences. Shiraz, Iran

a2 School of Biological Sciences, University of Liverpool, Crown St, Liverpool L69 7ZB

SUMMARY

A panel of microsatellites mapped to the Leishmania genome might make it possible to find associations between specific loci and phenotypic traits. To identify such loci, a Perl programme was written that scans the sequence of a genome and writes all loci containing microsatellites to a MySQL database. The programme was applied to the sequences of the L. braziliensis, L. infantum and L. major genomes. The database is publicly available over the internet: http://www.genomics.liv.ac.uk/tryps/resources.html ‘Microsatellite Locus Extractor’, and allows the selection of mapped microsatellites that meet user-defined criteria from a specified region of the selected genome. The website also incorporates a primer design pipeline that will design primers to amplify the selected loci. Using this pipeline 12 out of 17 primer sets designed against the L. infantum genome generated polymorphic PCR products. A tailed primer protocol was used to label all microsatellite primers with a single set of labelled primers. To avoid the culture of parasites prior to genotyping, sets of nested PCR primers were developed to amplify parasite DNA eluted from microscope slides. The limit of detection was approximately 1·6 parasite equivalents. However, only 6/56 DNA from slides stored at ambient temperature for over 6 months gave positive PCR results.

(Received November 09 2007)

(Revised January 04 2008)

(Accepted January 05 2008)

(Online publication March 27 2008)

Correspondence:

c1 Corresponding author: Room 231 Biosciences Building, University of Liverpool, Crown Street, Liverpool L69 7ZB. Tel: +0151 795 4512. www.genomics.liv.ac.uk/tryps. E-mail: harry@liv.ac.uk

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