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 inclusio n of the novel nucleotides, isoC and isoG, in the amplification sequen ces to prevent non-specific hybridization. The novel isoC, isoG-contai ning amplification sequences have no detectable interaction with any n atural DNA sequence, The control of non-specific hybridization in turn permits increased signal amplification, Addition of a 14 site preampl ifier 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 similar to 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.