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Enzymatic amplification of mini-exon-derived RNA gene spacers of Leishmania donovani: primers and probes for DNA diagnosis

Published online by Cambridge University Press:  06 April 2009

Q. Hassan ,
A. Ghosh ,
S. S. Ghosh ,
M. Gupta ,
D. Basu ,
K. K. Mallik  and
S. Adhya
Q. Hassan
Affiliation:
Genetic Engineering Laboratory (Leishmania Group), Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Calcutta 700032, India
A. Ghosh
Affiliation:
Genetic Engineering Laboratory (Leishmania Group), Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Calcutta 700032, India
S. S. Ghosh
Affiliation:
Genetic Engineering Laboratory (Leishmania Group), Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Calcutta 700032, India
M. Gupta
Affiliation:
Genetic Engineering Laboratory (Leishmania Group), Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Calcutta 700032, India
D. Basu
Affiliation:
School of Tropical Medicine, Calcutta, India
K. K. Mallik
Affiliation:
School of Tropical Medicine, Calcutta, India
S. Adhya
Affiliation:
Genetic Engineering Laboratory (Leishmania Group), Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Calcutta 700032, India
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Summary

The multicopy mini-exon-derived RNA (med RNA) locus of Leishmania donovani was enzymatically amplified by the polymerase chain reaction (PCR). The major 180 bp PCR product contained conserved med RNA gene sequences flanking the variable intergenic spacer from the med RNA gene tandem repeat. The oligonucleotide primers cross-reacted with other Leishmania species. In serial dilution experiments, positivity in the PCR assay was observed down to the genomic DNA equivalent of less than a single Leishmania cell. When the major PCR products from Indian L. donovani isolates were cloned and used as probes in dot hybridization analyses, they discriminated between L. donovani and L. amazonensis, L. major and L. infantum under high stringency conditions. DNA from spleen biopsies and blood samples of confirmed kala azar patients was positive, as were two skin biopsies from patients with post-kala azar dermal leishmaniasis (PKDL). These observations demonstrate that PCR amplification of med RNA intergenic spacers is sufficiently sensitive for clinical diagnosis of kala azar and PKDL, and furthermore, that cloned intergenic spacer probes may be useful for identification and classification of L. donovani.

Keywords

Leishmania donovanimed RNA genesPCRhybridization
Type
Research Article
Information
Parasitology , Volume 107 , Issue 5 , December 1993 , pp. 509 - 517
Copyright
Copyright © Cambridge University Press 1993

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References

REFERENCES

Avila, H. A., Sigman, D. S., Cohen, L. M., Millikan, R. C. & Simpson, L. (1991). Polymerase chain reaction amplification of Trypanosoma cruzi kinetoplast minicircle DNA isolated from whole blood lysates: Diagnosis of chronic Chagas' disease. Molecular and Biochemical Parasitology 48, 211–22.CrossRefGoogle ScholarPubMed
Barker, D. C. (1989). Molecular approaches to DNA diagnosis. Parasitology 99 (Suppl.), S125–146.CrossRefGoogle ScholarPubMed
Bhaumik, M., Das, S. & Adhya, S. (1991). Evidence for translational control of β-tubulin synthesis during differentiation of L. donovani. Parasitology 103, 197–205.CrossRefGoogle Scholar
De Bruijn, M. H. L. & Barker, D. C. (1992). Diagnosis of New World leishmaniasis: Specific detection of species of the Leishmania braziliensis complex by amplification of kinetoplast DNA. Acta Tropica 52, 45–58.CrossRefGoogle ScholarPubMed
De Franchis, R., Cross, N. C. P., Foulkes, N. S. & Cox, T. M. (1988). A potent inhibitor of Taq polymerase copurifies with human genomic DNA. Nucleic Acids Research 16, 10355.CrossRefGoogle ScholarPubMed
Evans, D. (1989). Handbook on Isolation, Characterization and Cryopreservation of Leishmania. pp. 28–30.Geneva: UNDP/World Bank/WHO.Google Scholar
Feinberg, A. P. & Vogelstein, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Analytical Biochemistry 132, 6–13.CrossRefGoogle ScholarPubMed
Hanahan, D. (1983). Techniques for transformation of E. coli. Journal of Molecular Biology 166, 557–80.CrossRefGoogle Scholar
Hassan, M. Q., Das, S. & Adhya, S. (1992). Mini-exon derived RNA gene of Leishmania donovani: Structure, organization and expression. Journal of Biosciences 17, 55–66.CrossRefGoogle Scholar
Jaffe, C. L., Grimaldi, G. & McMahon-Pratt, D. (1984). The cultivation and cloning of Leishmania. In Genes and Antigens of Parasites, a Laboratory Manual, 2nd Edn (ed. Morel, C. M.), pp. 47–91. Rio de Janeiro: Fundação Oswaldo Cruz.Google Scholar
Le Blancq, S. M. & Peters, W. (1986). Leishmania in the Old World: 4. The distribution of L. donovani sensu lato zymodemes. Transactions of the Royal Society of Tropical Medicine and Hygiene 80, 367–77.CrossRefGoogle ScholarPubMed
Leon, W., Fouts, D. L. & Manning, J. (1978). Sequence arrangement of the 16S and 26S rRNA genes in the pathogenic haemoflagellate Leishmania donovani. Nucleic Acids Research 5, 491–503.CrossRefGoogle Scholar
Maniatis, T., Fritsch, E. & Sambrook, J. (1982). In Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory.Google Scholar
Miller, S. I., Landfear, S. M. & Wirth, D. F. (1986). Cloning and characterization of a Leishmania gene encoding a RNA spliced leader sequence. Nucleic Acids Research 14, 7341–60.CrossRefGoogle ScholarPubMed
Pal, A., Mukherji, K., Basu, D., Naskar, K., Mallik, K. K. & Ghosh, D. K. (1991). Evaluation of a direct Leishmanja donovani med RNA gene spacers agglutination test (DAT) and ELISA for serodiagnosis of visceral leishmaniasis in India. Journal of Clinical Laboratory Analysis 5, 303–6.CrossRefGoogle 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 with a thermostable DNA polymerase. Science 239, 487–91.CrossRefGoogle ScholarPubMed
Simpson, L. (1987). The mitochondrial genome of kinetoplastid protozoa: genomic organization, transcription, replication and evolution. Annual Review of Microbiology 41, 363–82.CrossRefGoogle ScholarPubMed
Smyth, A. J., Ghosh, A., Hassan, M. Q., Basu, D., De Bruijn, M. H. L., Adhya, S., Mallik, K. K. & Barker, D. C. (1992). Rapid and sensitive detection of Leishmania kinetoplast DNA from spleen and blood samples of kala azar patients. Parasitology 105, 183–92.CrossRefGoogle ScholarPubMed
Wilson, K., Hanson, S., Landfear, S. & Ullman, B. (1991). Nucleotide sequences of the Leishmania donovani med RNA gene. Nucleic Acids Research 19, 5787.CrossRefGoogle Scholar
World Health Organization (1990). The Leishmaniases. WHO Technical Report Series no. 793, p. 154.Google Scholar