A One-Tube Quantitative HIV-1 RNA NASBA Nucleic Acid Amplification Assay Using Electrochemiluminescent (ECL) Labelled Probes

J Virol Methods. 1994 Sep;49(2):157-67. doi: 10.1016/0166-0934(94)90040-x.

Abstract

Quantification of HIV-1 viral RNA based on co-amplification of an internal standard Q-RNA dilution series requires a number of NASBA nucleic acid amplification reactions. For each internal standard Q-RNA concentration a separate NASBA amplification has to be performed. The development of an electrochemiluminescent (ECL) detection system with a dynamic signal detection range over 5 orders of magnitude enabled simplification of the Q-NASBA protocol. Three distinguishable Q-RNAs (QA, QB and QC) are mixed with the wild-type sample at different amounts (i.e. 10(4) QA, 10(3) QB and 10(2) QC molecules) and co-amplified with the wild-type RNA in one tube. Using ECL-labelled oligonucleotides the wild-type, QA, QB and QC amplificates are separately detected with a semi-automated ECL detection instrument and the ratio of the signals determined. The amount of initial wild-type RNA can be calculated from the ratio of wild-type signal to QA, QB and QC signals. This one-tube Q-NASBA protocol was compared to the earlier described protocol with six amplifications per quantification using model systems and HIV-1 RNA isolated from plasma of HIV-1-infected individuals. In all cases the quantification results of HIV-1 RNA were comparable between the two methods tested. Due to the use of only one amplification per quantification in the one-tube Q-NASBA protocol the QA, QB and QC internal standard RNA molecules can be added to the sample before nucleic acid isolation. The ratio of QA:QB:QC:WT RNAs, from which the initial input of WT-RNA is calculated, will remain constant independent of any loss that might occur during the nucleic acid isolation.

Publication types

  • Comparative Study

MeSH terms

  • Acquired Immunodeficiency Syndrome / blood*
  • Base Sequence
  • DNA Primers
  • HIV-1 / genetics
  • HIV-1 / isolation & purification*
  • Humans
  • Luminescent Measurements
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods*
  • RNA, Viral / analysis*
  • Reference Standards
  • Reproducibility of Results

Substances

  • DNA Primers
  • RNA, Viral