COMPARISON BETWEEN MICROWELL AND BEAD SUPPORTS FOR THE DETECTION OF HUMAN CYTOMEGALOVIRUS AMPLICONS BY SANDWICH HYBRIDIZATION

In this study, we compared the efficiency of capture DNA probes covale ntly bound onto magnetic beads or microplates for their hybridization with target human cytomegalovirus (HCMV) DNA amplicons. Polystyrene su pports were first aminated by wet chemistry to allow covalent grafting of the capture probes. The level of amines grafted was three times hi gher on beads than on microwells. Increasingly higher sizes of capture probes were fixed on both supports and the best reaction yield ranged from 300 to 500 fmol. The sizes of the capture and detection probes w ere optimized in order to obtain high target DNA hybridization yield. Long capture probes were more accessible than short ones to the target , with faster kinetics of hybridization obtained on beads than on micr oplates. Sensitivity of the hybridization assay was then determined wi th a nonisotopic method and the detection limit found was 30 amol of H CMV amplicons on both supports. HCMV DNA extracted from clinical sampl es were amplified by PCR. The resulting amplicons were then analyzed u sing the optimized sandwich hybridization assay discussed here. The re sults perfectly fitted with the qualitative conclusions obtained after a nested PCR analyzed on agarose gel. (C) 1997 Academic Press.