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

• Published in: Nucleic acids research Vol. 25; no. 15; pp. 2979 - 2984
• Main Authors: Collins, Mark L., Irvine, Bruce, Tyner, Diana, Fine, Eric, Zayati, Crystle, Chang, Chu-an, Horn, Thomas, Ahle, David, Detmer, Jill, Shen, Lu-Ping, Kolberg, Janice, Bushnell, Steve, Urdea, Mickey S., Ho, David D.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.08.1997
• Subjects: AIDS/HIV; Anti-HIV Agents - therapeutic use; Cytidine - chemistry; DNA - chemistry; DNA - metabolism; DNA, Viral - analysis; Drug Therapy, Combination; Guanosine - chemistry; HIV; HIV Infections - drug therapy; HIV Infections - virology; Humans; Isoquinolines - therapeutic use; Lamivudine - therapeutic use; Nelfinavir; Nucleic Acid Hybridization - methods; RNA, Viral - analysis; Sulfonic Acids - therapeutic use; Zidovudine - therapeutic use
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/25.15.2979

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 ∼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.