Hostname: page-component-77f85d65b8-6c7dr Total loading time: 0 Render date: 2026-04-12T16:45:55.441Z Has data issue: false hasContentIssue false
  • English
  • Français

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
Get access Rights & Permissions [Opens in a new window]

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

Information

Type
Research Article
Information
Parasitology , Volume 105 , Issue 2 , October 1992 , pp. 183 - 192
Copyright
Copyright © Cambridge University Press 1992

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Article purchase

Temporarily unavailable

References

REFERENCES

Barker, D. C., Gibson, L. J., Kennedy, W. P. K., Nasser, A. A. A. A. & Williams, R. H. (1986). The potential of using recombinant DNA species specific probes for the identification of tropical Leishmania. Parasitology 91 (Suppl.), S139–S174.CrossRefGoogle Scholar
Borst, P. & Fairlamb, A. H. (1976). DNA of parasites, with special reference to kinetoplast DNA. In Biochemistry of Parasites and Host-Parasite Relationships (ed. Van den Bossche, H.), pp. 169–92. Amsterdam: North Holland Publishing Co.Google Scholar
Borst, P. & Hoeijmakers, J. H. J. (1979). Kinetoplast DNA: a review. Plasmid 2, 20–40.CrossRefGoogle Scholar
Chaudhury, P. K. & Kundu, B. (1987). Splenectomy in drug resistant Kala-azar. In Visceral Leishmaniasis and Post-Kala-Azar Dermal Leishmaniasis, 1st Edn, (ed. Mallik, K. K.), pp. 69–73. Calcutta: Calcutta School of Tropical Medicine.Google Scholar
De Beer, P., El Harith, A., Grootheest, M. & Winkler, A. (1990). Outbreak of kala-azar in the Sudan. Lancet 335, 224.CrossRefGoogle ScholarPubMed
De Bruijn, M. H. L. (1984). Diagnostic DNA amplification: no respite for the elusive parasite. Parasitology Today 4, 293–5.CrossRefGoogle Scholar
Jayaraman, K. S. (1988). Leishmaniasis resurgent in India. Nature, London 333, 590.Google ScholarPubMed
Kennedy, W. P. K. (1984). Novel identification of differences in the kinetoplast DNA of Leishmania isolates by recombinant DNA techniques and in situ hybridization. Molecular and Biochemical Parasitology 12, 313–25.Google Scholar
Locksley, R. M. (1991). Leishmaniasis. In Harrisons's Principles of Internal Medicine, vol. 1, 13th Edn., (ed. Wilson, J. D., Braunwald, E. & Isselbacher, K. J.), pp. 789–91. New York: McGraw-Hill.Google Scholar
Mallik, K. K., Basu, D. & Mondal, N. K. (1987). Management of sodium antimony gluconate failure cases of visceral leishmaniasis. In Visceral Leishmaniasis and Post-Kala-Azar Dermal Leishmaniasis, 1st Edn., (ed. Mallik, K. K.), pp. 65–68. Calcutta: School of Tropical Medicine.Google Scholar
Messing, J. & Vieira, J. (1982). The pUC plasmids, a M13mp7 derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259–68.Google Scholar
Rodgers, M. R., Popper, S. J. & Wirth, D. F. (1990). Amplification of kinetoplast DNA as a tool in the detection and diagnosis of Leishmania. Experimental Parasitology 71, 267–75.CrossRefGoogle ScholarPubMed
Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B. & Ehrlich, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–91.CrossRefGoogle ScholarPubMed
Sattaur, O. (1991). Doubts over testing hamper India's AIDS effort. New Scientist, no. 1765, p. 18.Google Scholar
Smith, D. F., Searle, S., Ready, P. D., Gramiccia, M. & Ben-Ismail, R. (1989). A kinetoplast DNA probe diagnostic for Leishmania major: sequence homologies between regions of Leishmania minicircles. Molecular and Biochemical Parasitology 37, 213–24.CrossRefGoogle ScholarPubMed
Staden, R. (1982). Automation of the computer handling of gel reading data produced by the shotgun method of DNA sequencing. Nucleic Acids Research 10, 4731–51.CrossRefGoogle ScholarPubMed
Stuart, K. (1983). Kinetoplast DNA, mitochondrial DNA with a difference. Molecular and Biochemical Parasitology 9, 93–104.CrossRefGoogle ScholarPubMed
Sturm, N. R., Degrave, W., Morel, C. M. M. & Simpson, L. (1989). Sensitive detection and schizodeme identification of Trypanosoma cruzi cells by amplification of kinetoplast DNA minicircle DNA sequences: Use in diagnosis of Chaga's disease. Molecular and Biochemical Parasitology 33, 205–14.CrossRefGoogle Scholar
World Health Organization (1990). The Leishmaniases. WHO Technical Report Series, no. 793, p. 154.Google Scholar