Adaptation of a radioactive L. donovani complex DNA probe to a chemiluminescent detection system gives enhanced sensitivity for diagnostic and epidemiological applications.
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 10(4) L. major. This cross-reaction was reduced by hybridizations in 50% formamide at 37 degrees 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.
Wilson
S M
SM
Department of Clinical Sciences, London School of Hygiene and Tropical Medicine.
McNerney
R
R
Moreno
M B
MB
Frame
I
I
Miles
M A
MA
D000818:Animals; D001345:Autoradiography; D015342:DNA Probes; D016054:DNA, Protozoan / Q000032:analysis*; D006801:Humans; D007893:Leishmania donovani / Q000235:genetics / Q000302:isolation & purification*; D007898:Leishmaniasis, Visceral / Q000175:diagnosis* / Q000453:epidemiology; D009693:Nucleic Acid Hybridization; D012680:Sensitivity and Specificity; D013045:Species Specificity
D015342:DNA Probes; D016054:DNA, Protozoan