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Adaptation of a radioactive L. donovani complex DNA probe to a chemiluminescent detection system gives enhanced sensitivity for diagnostic and epidemiological applications

Published online by Cambridge University Press:  06 April 2009

S. M. Wilson ,
R. Mcnerney ,
M. B. Moreno ,
I. Frame  and
M. A. Miles
S. M. Wilson
Affiliation:
Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT
R. Mcnerney
Affiliation:
Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT
M. B. Moreno
Affiliation:
Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT
I. Frame
Affiliation:
Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT
M. A. Miles
Affiliation:
Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT
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Extract

The cDNA probe, Lmet2, was labelled with digoxigenin and used in a chemiluminescent system to detect fewer than 100 membrane-immobilized Leishmania parasites. The probe was found to hybridize primarily with members of the L. donovani complex but a slight cross-reaction was also observed with greater than 5 x 104L. major. This cross-reaction was reduced by hybridizations in 50% formamide at 37 °C. Formamide also significantly reduced non-specific binding of the digoxigenin-labelled probe to the membrane support which, in hybridizations without formamide, masked the specific hybridization signal. This background was not observed with the corresponding radio-isotope labelled probe. With hybridizations in formamide the sensitivity achieved by the chemiluminescent system after exposure to film for 3 h was greater than that achieved by the isotopic system even after autoradiography for 24 h.

Keywords

LeishmaniaDNA probeshybridizationchemiluminescence
Type
Research Article
Information
Parasitology , Volume 104 , Issue 3 , June 1992 , pp. 421 - 426
Copyright
Copyright © Cambridge University Press 1992

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