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

Published online by Cambridge University Press:  06 April 2009

A. J. Smyth ,
A. Ghosh ,
Md. Q. Hassan ,
D. Basu ,
M. H. L. De Bruijn ,
S. Adhya ,
K. K. Mallik  and
D. C. Barker
A. J. Smyth
Affiliation:
MRC Outstation of NIMR, Molteno Laboratories, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1 QP, UK
A. Ghosh
Affiliation:
Genetic Engineering Laboratory, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Calcutta 700 032, India
Md. Q. Hassan
Affiliation:
Genetic Engineering Laboratory, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Calcutta 700 032, India
D. Basu
Affiliation:
Department of Tropical Medicine, School of Tropical Medicine, Chittaranjan Avenue, Calcutta 700 073, India
M. H. L. De Bruijn
Affiliation:
MRC Outstation of NIMR, Molteno Laboratories, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1 QP, UK
S. Adhya
Affiliation:
Genetic Engineering Laboratory, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Calcutta 700 032, India
K. K. Mallik
Affiliation:
Department of Tropical Medicine, School of Tropical Medicine, Chittaranjan Avenue, Calcutta 700 073, India
D. C. Barker
Affiliation:
MRC Outstation of NIMR, Molteno Laboratories, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1 QP, UK
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Summary

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.

Keywords

Leishmania donovanikinetoplast DNAkala-azarpolymerase chain reaction
Type
Research Article
Information
Parasitology , Volume 105 , Issue 2 , October 1992 , pp. 183 - 192
Copyright
Copyright © Cambridge University Press 1992

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