Parasitology

Research Article

Rapid and sensitive detection of Leishmania kinetoplast DNA from spleen and blood samples of kala-azar patients

A. J. Smytha1, A. Ghosha2, Md. Q. Hassana2, D. Basua3, M. H. L. De Bruijna1, S. Adhyaa2, K. K. Mallika3 and D. C. Barkera1*

a1 MRC Outstation of NIMR, Molteno Laboratories, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1 QP, UK

a2 Genetic Engineering Laboratory, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Calcutta 700 032, India

a3 Department of Tropical Medicine, School of Tropical Medicine, Chittaranjan Avenue, Calcutta 700 073, India

Abstract

Following sequence analysis of a Leishmania donovani kinetoplast DNA (kDNA) minicircle, we have developed synthetic oligonucleotides for use in the polymerase chain reaction (PCR). With these primers, we have amplified L. donovani kDNA from splenic aspirates and blood samples taken from kala-azar patients. Treatment of the samples for PCR requires only limited DNA purification by lysis in SDS, digestion with proteinase K, phenol extraction and ethanol precipitation of the resulting nucleic acid. We have obtained amplified product routinely with DNA prepared from the equivalent of 2·5–25 µl of splenic aspirate or of 50–500 µl of blood from infected patients. In dilution experiments a visible product has been obtained on amplification of DNA from the equivalent of 2·5 × 10−7 µl of splenic material. We therefore propose the amplification of L. donovani kDNA by PCR as a rapid and highly sensitive method for the diagnosis of kala-azar.

(Received September 24 1991)

(Revised February 10 1992)

(Accepted February 12 1992)

Footnotes

* Reprint requests to Dr D. C. Barker.

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