Detection and identification of Leishmania DNA within naturally infected sand flies by seminested PCR on minicircle kinetoplastic DNA

A seminested PCR assay was developed in order to amplify the kinetoplast mi nicircle of Leishmania species from individual sand flies. The kinetoplast minicircle is an ideal target because it is present in 10,000 copies per ce ll and its sequence is known for most Leishmania species. The two-step PCR is carried out in a single tube using three primers, which were designed wi thin the conserved area of the minicircle and contain conserved sequence bl ocks. The assay was able to detect as few as 3 parasites per individual san d fly and to amplify minicircle DNA from at least eight Leishmania species. This technique permits the processing of a large number of samples synchro nously, as required for epidemiological studies, in order to study infectio n rates in sand fly populations and to identify potential insect vectors. C omparison of the sequences obtained from sand flies and mammal hosts will b e crucial for developing hypotheses about the transmission cycles of leishm ania spp. in areas of endemicity.