Development of a real-time quantitative RT-PCR for the detection of HIV-2 RNA in plasma

Martin Schutten, B van den Hoogen, Marchina E. van der Ende, R A Gruters, A D Osterhaus, Bert Niesters

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Abstract

An assay is described for the quantification of human immunodeficiency virus type 2 (HIV-2) RNA in EDTA plasma based on RT-PCR using the Taqman real-time PCR detection method. As standard, an electron microscopically counted virus stock of HIV-2 strain NIHZ was used. The lower detection limit is 5 # 102 HIV-2 RNA copies per ml of EDTA plasma. The assay is linear within the range required (5 # 102-106 HIV-2 RNA copies/ml of EDTA plasma) with an intra assay variability of 2.5% and an inter-assay variability ranging from 2% at 106 copies to 7.5% at the lower detection limit. Three primer/probe combinations were developed to circumvent false negative samples due to nucleotide variation in the target sequence. Using these primer/probe sets enabled the detection of HIV-2 DNA sequences from all HIV-2 seropositive individuals and two out of five dual human immunodeficiency virus type 1 (HIV-1) and HIV-2 seropositive individuals visiting the University Hospital Rotterdam.

Original languageEnglish
Pages (from-to)81-87
Number of pages7
JournalJournal of virological methods
Volume88
Issue number1
Publication statusPublished - Jul-2000
Externally publishedYes

Keywords

  • HIV Infections
  • HIV-1
  • HIV-2
  • Humans
  • RNA, Viral
  • Reverse Transcriptase Polymerase Chain Reaction
  • Taq Polymerase
  • Viral Load

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