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 st ep is often performed but this usually involves an entirely new proced ure with additional manipulations, expense and time. We have simplifie d this post-PCR hybridization by developing a strategy which includes the probe in the PCR and enables the hybridization to be performed aut omatically as part of the PCR programme. The hybrids are afterwards de tected by capture in microtitre wells and colorimetric visualization. This method, which we have termed PCR-solution hybridization enzyme-li nked immunoassay (PCR-SHELA), is rapid, able to detect less than 5 cul tured parasites and is specific for parasites of the Leishmania donova ni-complex. We also describe the application of PCR-SHELA to the detec tion of amastigotes in various tissues of infected laboratory animals.