Screening and Characterization of RAPD Markers in Viscerotropic Leishmania Parasites

Abstract

Visceral leishmaniasis (VL) is mainly due to the Leishmania donovani complex. VL is endemic in many countries worldwide including East Africa and the Mediterranean region where the epidemiology is complex. Taxonomy of these pathogens is under controversy but there is a correlation between their genetic diversity and geographical origin. With steady increase in genome knowledge, RAPD is still a useful approach to identify and characterize novel DNA markers. Our aim was to identify and characterize polymorphic DNA markers in VL Leishmania parasites in diverse geographic regions using RAPD in order to constitute a pool of PCR targets having the potential to differentiate among the VL parasites. 100 different oligonucleotide decamers having arbitrary DNA sequences were screened for reproducible amplification and a selection of 28 was used to amplify DNA from 12 L. donovani, L. archibaldi and L. infantum strains having diverse origins. A total of 155 bands were amplified of which 60.65% appeared polymorphic. 7 out of 28 primers provided monomorphic patterns. Phenetic analysis allowed clustering the parasites according to their geographical origin. Differentially amplified bands were selected, among them 22 RAPD products were successfully cloned and sequenced. Bioinformatic analysis allowed mapping of the markers and sequences and priming sites analysis. This study was complemented with Southern-blot to confirm assignment of markers to the kDNA. The bioinformatic analysis identified 16 nuclear and 3 minicircle markers. Analysis of these markers highlighted polymorphisms at RAPD priming sites with mainly 5′ end transversions, and presence of inter– and intra– taxonomic complex sequence and microsatellites variations; a bias in transitions over transversions and indels between the different sequences compared is observed, which is however less marked between L. infantum and L. donovani. The study delivers a pool of well-documented polymorphic DNA markers, to develop molecular diagnostics assays to characterize and differentiate VL causing agents.

Molecular Tools for Understanding Eco-Epidemiology, Diversity and Pathogenesis of Leishmania Parasites

“…These primers organized the parasites according to their geographical origin in a similar way to other studies using RAPD or other types of tools [39,41]. Some of the differentially amplified RAPD bands obtained in our study were cloned and sequenced; their analysis with bioinformatics tools and comparison to their respective genomic sites in L. infantum, L. donovani and L. major genomes highlighted the markers' association with simple sequence repeats and microsatellites in non coding regions [51]. A selection of such markers in L. archibaldi was used to develop simple PCR assays differentiating viscerotropic parasites, some of which in a country-specific way [52].…”

“…Alternatively, with the objective to identify markers and develop simple assays for the discrimination of viscerotropic parasites encountered in Africa, we have screened 5 Operon kits (100 primers) for reproducible profiles and a selection of 28 primers was then used to screen for DNA markers within a panel of viscerotropic parasites from different countries in Africa and India [51]. These primers organized the parasites according to their geographical origin in a similar way to other studies using RAPD or other types of tools [39,41].…”

“…Although simple and having potential for detecting variation where other techniques fail, other drawbacks of this technique could be that bands of equal electrophoretic mobility may not be homologous [34]; identification of allelic variants is also not possible in Leishmania. Yet, RAPD constitutes a powerful tool for the identification of markers and the design of PCR based assays [34,[49][50][51][52]. Diverse RAPD Leishmania studies reached conclusions that were confirmed by other tools or alternative studies making the RAPD approach still valuable.…”

Genetic Diversity Analysis of Chinese Leishmania Isolates and Development of L. donovani Complex Specific Markers by RAPD

“…As we all know, RAPD has its innate drawbacks in stability and repeatability of bands due to the highly random hybrid sites with template DNA. Therefore, to gain stable bands and repeatable results of RAPD, we followed the PCR ampli ed condition whose stability and repeatability has been already reported [27,28]and had validated the results before formal experiments. Meanwhile, the usage of commercial Taq DNA Mix from the same batch replaced the addition of dNTP, Mg 2+ and Taq DNA polymerase one by one also improved the reaction stability in this study.…”

“…Twenty decamer primers were selected according to previous studies [27,28]and commercially synthesized (Invitrogen). All primers were prepared as 10 µM (10 pmol/µl) working solutions.…”