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Applied and Environmental Microbiology, May 2000, p. 1933-1938, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Detection and Identification of Leishmania DNA within Naturally Infected Sand Flies by Seminested PCR on Minicircle Kinetoplastic DNA

Ana M. Aransay,* Efstathia Scoulica, and Yannis Tselentis

Laboratory of Clinical Bacteriology, Parasitology, Zoonoses, and Geographical Medicine (WHO Collaborating Centre for Research and Training in Mediterranean Zoonoses), Faculty of Medicine, University of Crete, Heraklion, Greece

Received 5 November 1999/Accepted 16 February 2000

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.

* Corresponding author. Mailing address: Molecular Systematics Laboratory, Entomology Department, The Natural History Museum, Cromwell Rd., London SW7 5BD, United Kingdom. Phone: 44 (0) 207 942 5866 or -5483. Fax: 44 (0) 207 942 5229. E-mail: A.Aransay{at}nhm.ac.uk.

Applied and Environmental Microbiology, May 2000, p. 1933-1938, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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