Detection of parasites of the Leishmania donovani-complex by a polymerase chain reaction-solution hybridization enzyme-linked immunoassay (PCR-SHELA).
A polymerase chain reaction (PCR) based on the detection of the Lmet2 repeat sequence specific to members of the Leishmania donovani-complex is described. To improve PCR specificity, a post-PCR hybridization step is often performed but this usually involves an entirely new procedure with additional manipulations, expense and time. We have simplified this post-PCR hybridization by developing a strategy which includes the probe in the PCR and enables the hybridization to be performed automatically as part of the PCR programme. The hybrids are afterwards detected by capture in microtitre wells and colorimetric visualization. This method, which we have termed PCR-solution hybridization enzyme-linked immunoassay (PCR-SHELA), is rapid, able to detect less than 5 cultured parasites and is specific for parasites of the Leishmania donovani-complex. We also describe the application of PCR-SHELA to the detection of amastigotes in various tissues of infected laboratory animals.
Qiao
Z
Z
Department of Epidemiology and Population Sciences, London School of Hygiene and Tropical Medicine, UK.
Miles
M A
MA
Wilson
S M
SM
D000818:Animals; D001483:Base Sequence; D015342:DNA Probes; D016054:DNA, Protozoan / Q000032:analysis* / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007893:Leishmania donovani / Q000235:genetics / Q000302:isolation & purification*; D007898:Leishmaniasis, Visceral / Q000175:diagnosis; D008099:Liver / Q000469:parasitology; D051379:Mice; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid; D012680:Sensitivity and Specificity; D013154:Spleen / Q000469:parasitology
D015342:DNA Probes; D016054:DNA, Protozoan