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. 2000 May;66(5):1933-8.
doi: 10.1128/AEM.66.5.1933-1938.2000.

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

A M Aransay  1 , E Scoulica, Y Tselentis
Affiliations

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

A M Aransay et al. Appl Environ Microbiol. 2000 May.

Abstract

A seminested PCR assay was developed in order to amplify the kinetoplast minicircle of Leishmania species from individual sand flies. The kinetoplast minicircle is an ideal target because it is present in 10,000 copies per cell 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 within the conserved area of the minicircle and contain conserved sequence blocks. The assay was able to detect as few as 3 parasites per individual sand fly and to amplify minicircle DNA from at least eight Leishmania species. This technique permits the processing of a large number of samples synchronously, as required for epidemiological studies, in order to study infection rates in sand fly populations and to identify potential insect vectors. Comparison of the sequences obtained from sand flies and mammal hosts will be crucial for developing hypotheses about the transmission cycles of Leishmania spp. in areas of endemicity.

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Figures

FIG. 1
FIG. 1
Map of the greater Athens area. Black circles show the areas where sand flies were collected for the present study.
FIG. 2
FIG. 2
Sensitivity comparison between standard PCR with primers LINR4 and LIN17 or LINR4 and LIN19 (A) and seminested PCR with the set of primers LINR4, LIN17, and LIN19 (B). The DNA size marker (lane M) is PUC19 TaqI/PUC19 Sau3A.
FIG. 3
FIG. 3
Specificity of the seminested PCR assay with primers LINR4, LIN17, and LIN19. The DNA size marker (lane M) is PUC19 TaqI/PUC19 Sau3A.
FIG. 4
FIG. 4
Seminested PCR with primers LINR4, LIN17, and LIN19 for detection of Leishmania DNA within sand flies collected in parts of the greater Athens area. − control, reaction without DNA; sf. male, male sandfly specimen; M, DNA size marker PUC19 TaqI/PUC19 Sau3A; P.pa, P. (Phlebotomus) papatasi; P.si, P. (Adlerius) simici; P.to, P. (Larroussius) tobbi; P.al, P. (Paraphlebotomus) alexandri; P.ne, P. (Larroussius) neglectus; + control, L. infantum (LEM 235).
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