A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml.

The branched DNA hybridization assay has been improved by the inclusion of the novel nucleotides, isoC and isoG, in the amplification sequences to prevent non-specific hybridization. The novel isoC, isoG-containing amplification sequences have no detectable interaction with any natural DNA sequence. The control of non-specific hybridization in turn permits increased signal amplification. Addition of a 14 site preamplifier was found to increase the signal/noise ratio 8-fold. A set of 74 oligonucleotide probes was designed to the consensus HIV POL sequence. The detection limit of this new HIV branched DNA amplifier assay was approximately 50 molecules/ml. The assay was used to measure viral load in 87 plasma samples of HIV- infected patients on triple drug therapy whose RNA titers were <500 molecules/ml. In all 11 patients viral load eventually declined to below the detection limit with the new assay.

Branched DNA amplification multimers for the sensitive, direct detection of human hepatitis viruses.

Branched oligonucleotides (bDNA) have been synthesized containing a unique primary segment and a set of identical secondary fragments covalently attached to the primary sequence through branch points. The primary sequence is designed to hybridize (directly or indirectly) to a target nucleic acid, such as hepatitis B virus (HBV) or hepatitis C virus (HCV) genomic DNA or RNA, respectively. The secondary fragments are used to direct the binding of multiple copies of a small oligonucleotide labelled with alkaline phosphatase. Assays for the presence of HBV and HCV based on the application of these branched amplification multimers have been devised. It is possible to detect as few as 1,000 hepatitis viral genomes directly.