A novel internally controlled real-time reverse transcription-PCR assay for HIV-1 RNA targeting the pol integrase genomic region

J Virol Methods. 2007 Jun;142(1-2):127-35. doi: 10.1016/j.jviromet.2007.01.013. Epub 2007 Feb 26.


Given the worldwide increasing spread of HIV-1 genetic variants, it is mandatory that assays used for nucleic acid testing for HIV-1 detect all existing groups and subtypes of HIV-1. In this report the development and evaluation of a quantitative real-time HIV-1 RT-PCR assay that targets a conserved region within the pol integrase domain is described. As an internal control reaction, endogenous glyceraldehyde-3-phosphate-dehydrogenase transcripts were detected in a multiplex configuration. The detection limit (95% cut-off value) was determined by probit analysis and calculated as 281 IU/ml of HIV-1 RNA. Within-run and between-run coefficients of variation were below 15 and 27%, respectively, indicating high reproducibility. The described assay detected all tested HIV-1 isolates representing groups M, O and N. Within group M, quantitative test results correlated well with viral loads as determined by the automated Abbott RealTime HIV-1 assay. Based on the testing of 1206 confirmed HIV-1 RNA negative blood donor samples, assay specificity was found to be 100%. The rate of inhibition was 0.37%. The described HIV-1 real-time RT-PCR was validated according to regulatory guidelines and is applicable to the screening of blood donors as well as the determination of HIV-1 viral load.

Publication types

  • Evaluation Study

MeSH terms

  • Blood Donors
  • Gene Products, pol / genetics*
  • Genetic Variation
  • Genome, Viral
  • Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / metabolism*
  • HIV Infections / diagnosis
  • HIV Infections / virology
  • HIV Integrase / genetics*
  • HIV-1 / classification
  • HIV-1 / genetics
  • HIV-1 / isolation & purification*
  • Humans
  • RNA, Viral / blood*
  • Reference Standards
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity


  • Gene Products, pol
  • RNA, Viral
  • Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)
  • HIV Integrase