Fast and sensitive quantitative detection of HIV DNA in whole blood leucocytes by SYBR green I real-time PCR assay

Anna Casabianca, Caterina Gori, Chiara Orlandi, Federica Forbici, Carlo Federico Perno, Mauro Magnani

Research output: Contribution to journalArticlepeer-review

Abstract

The aim of this study was the development of a real-time PCR for HIV DNA quantification in whole blood leucocytes providing an alternative assay to those already described, almost based on the gag gene detection. The technique (pbs-rtPCR assay) is more rapid (the whole assay required less than 5 h), easy to perform, omitting both PBMC purification step and data normalization to a housekeeping gene, when compared to previously published assays. Our method is able to detect all group M HIV-1 subtypes in the highly conserved primer-binding site (PBS) region and to avoid the interference by retroviral endogenous sequences in HIV DNA quantification. The sensitivity was 100% for 2 copies even in the presence of high amounts of genomic DNA (1 μg). To monitor the HIV DNA level in all possible clinical conditions, the assay has been validated and compared with a previously developed gag-PCR assay on 73 HIV-1 HAART-treated patients with a plasma HIV-1 RNA range from 500 000 copies/ml. The 50% of the samples with a viremia below the limit of detection (50 copies/ml) was found to contain HIV DNA between 2 and 10 copies/μg DNA. The pbs-rtPCR offers a significant improvement in the percentage of quantitatively detectable sample (99%) compared with the gag-PCR (42%) suggesting caution in the choice of HIV DNA assay.

Original languageEnglish
Pages (from-to)368-378
Number of pages11
JournalMolecular and Cellular Probes
Volume21
Issue number5-6
DOIs
Publication statusPublished - Oct 2007

Keywords

  • HIV DNA quantification
  • Human endogenous retroviruses
  • Primer-binding site
  • SYBR Green I real-time PCR
  • Whole blood leucocytes

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology

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