DIRECT QUANTITATION OF HIV BY FLOW-CYTOMETRY USING BRANCHED DNA SIGNAL AMPLIFICATION

Adaptation of the branched DNA signal amplification technology to flow cytometry has resulted in a quantitative nucleic-acid assay with sign ificant advantages over the microwell-based format. In this assay, mic robeads, rather than microwell plates, are derivatized with nucleic-ac id capture probes and the derivatized beads are used to capture single nucleic-acid targets, which then capture fluorescent reporter probes via branched DNA. The assay detects DNA or RNA targets, has a current lower sensitivity limit of 500 human immunodeficiency virus (HIV) RNA molecules and responds linearly to target level from 500 to at least 5 0 000 molecules. Since microbeads can easily interrogate large volumes , viral lysis and genomic RNA capture can proceed in one step from com paratively large volumes, and sample preparation is greatly simplified compared to the microwell-format bDNA assay. (C) 1998 Academic Press.