Molecular analyses of Old World Leishmania RAPD markers and development of a PCR assay selective for parasites of the L. donovani species Complex.

Three amplicons, appearing in a species-specific manner on the electrophoregrams of RAPD reactions that were obtained with primer OPA1, OPA1-800, OPA1- 900, and OPA1-1200, are analyzed in this study. The study revealed that each of these products is composed of one Leishmania DNA band, taxonomically conserved among the different Old World species studied. Subsequently, only the electrophoretic position of the RAPD products can be considered species-specific. In addition, sequence data, genomic organization, and chromosomal location have proved that these fragments are different and physically independent. However, they possess common features related to the presence of different kinds of short DNA repeats, more particularly microsatellites and a CCCTTC motive, corresponding to the 3' half of the OPA1 primer. These results suggest that the OPA1 primer has initiated amplification from different priming sites, having a species-specific location. This corresponds to sequence micro-heterogeneity of DNA fragments present within the different species and leading eventually to a selective amplification of different RAPD products. This characteristic has been used to develop an original selective PCR test based on the sequence of the OPA1-800 product, in which only DNAs from the L. donovani species complex are amplified. Restriction site polymorphisms and sequence variations are identified within the PCR fragment amplified from these parasite DNAs. In fact, the OPA1-800 fragment proved to be a useful DNA marker either as a DNA probe or as a target for PCR-based assays. This tool can therefore be recommended for the control of Old World Leishmania parasites, such as species discrimination, molecular tracking of isolates, or study of polymorphisms within the L. donovani species complex. Moreover, the molecular bases underlying the amplification of the RAPD fragments studied correspond to mechanisms already described. Although they do not account for the amplification of all Leishmania RAPD products, such mechanisms stress some of the pitfalls of the technique, which need to be taken into consideration. We have identified at least misleading observations of DNA bands amplified in a species-specific manner, in spite of their presence in the genome of the other taxa, and relatedness between bands within the amplification profiles. Therefore, recommendations for careful interpretation of RAPD data in population genetics or phylogenetic analyses are reiterated. Molecular analyses are essential to validate conclusions.