Quantification of HIV-1 RNA Using a Homogeneous Single-Tube Assay

[摘要] Quantification of HIV RNA has become an essential tool in the management of HIV infection and monitoring of gene expression. Although direct quantification of nucleic acids has been demonstrated, the principal method of quantifying nucleic acids still involves nucleic acid amplification and quantification of the specific amplification products. Most commonly, quantification of nucleic acids involves either real-time monitoring of the amplification products [with fluorescently labeled probes (TaqMan; Molecular Beacons)] or postamplification quantification [e.g., electrochemiluminescence (Nuclisens™)] (1)(2)(3). Real-time monitoring increases the dynamic range of quantification, but it also leads to more complex instrumentation, and the absence of an internal control in the reaction may lead to erroneous quantification because of sample interference. Quantitative nucleic acid sequence-based amplification (NASBA™) is carried out using internal controls added to the sample, but the presence of three internal controls and a postamplification electrochemiluminescence detection scheme makes this method susceptible to contamination of samples with previously amplified products.We have combined highly sensitive NASBA with a highly sensitive chemiluminescent detection technology, Luminescent Oxygen Channeling Immunoassay (LOCI™), for homogeneous quantification of nucleic acids. NASBA is an isothermal exponential nucleic acid amplification system, based on an activity of avian myeloblastosis virus reverse transcriptase, RNase H, and T7 RNA polymerase, which predominantly produces a single-stranded RNA product. The specificity of HIV-1 amplification and detection is ensured by the use of specific primers and is further enhanced by the use of specific probe sequences for detection. The product concentration can be as high as 8.0 μmol/L after a typical NASBA reaction. Because the amplification product is single-stranded, there is no need to denature at the end of amplification for the binding of specific probes for either detection or quantification. Addition of an internal control (Qa), which bears sequence resemblance and is coamplified with the same primers …

[学科分类] 过敏症与临床免疫学