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 perform 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 tissues of infected laboratory animals