Quantification of HIV-1 proviral DNA from peripheral blood mononuclear cells using a high throughput four-competitor competitive PCR

Ubaldo Visco Comandini, Anders Sönnerborg, Anders Vahlne, Zhibing Yun

Research output: Contribution to journalArticlepeer-review

Abstract

A multiple competitor PCR (mcPCR) was developed to quantify HIV-1 proviral DNA from peripheral blood mononuclear cells (PBMC). DNA extracted from a mixture of HIV infected PBMC and four size-mutated DNA competitors were co-amplified. The Cy5-fluorescence labelled PCR products were denatured by heating, separated using an automated DNA sequencer and quantified by a fragment analysis computer software. An internal standard was generated by plotting the peak areas of the four competitors against their inputs. Based on the internal standard, HIV sample DNA was quantified by extrapolating the corresponding signal. The linear range of the mcPCR was three log wide and the quantitation limit was about 20 copies of HIV DNA/10 6 PBMC. Using the mcPCR, HIV DNA was quantified from 14 long-term non progressors (LTNP) and 14 patients with advanced disease. A significantly lower copy number of HIV DNA was obtained in the LTNP (p = 0.018). These data suggest that the mcPCR is sensitive, reliable and especially useful for HIV DNA quantification of a large number of clinical samples.

Original languageEnglish
Pages (from-to)171-180
Number of pages10
JournalJournal of Virological Methods
Volume69
Issue number1-2
DOIs
Publication statusPublished - Dec 1997

Keywords

  • DNA quantification
  • Fluorescent detection
  • HIV-1
  • Long-term non progressors
  • Multiple competitors PCR

ASJC Scopus subject areas

  • Virology

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