Quantitative recovery of proviral HIV-1 DNA from leukocytes by the Dried Buffy Coat Spot method for real-time PCR determination

Marco Rossi de Gasperis, Maria Daniela Caione, Carlo Concato, Ersilia Fiscarelli, Nicoló Di Pietro, Vittorio Salotti, Lorenza Putignani, Donato Menichella, Francesco Callea

Research output: Contribution to journalArticlepeer-review

Abstract

The current recommended method for diagnosing HIV-1 in newborns infected vertically and in adults, during the "window period", is the detection of proviral HIV-1 DNA within leukocytes (buffy coat). This study describes a new portable Dried Buffy Coat Spot (DBCS) assay able to provide a quantitative proviral HIV-1 DNA recovery from the buffy coat. Fifty blood samples were collected from HIV-positive children and processed for DBCSs. Total DNA and proviral DNA were normalised to β-globin and HIV-1 pol genes. Assay sensitivity and specificity were evaluated against the whole blood dried blood spot (DBS) method. Both procedures, using automatic DNA extraction, were compared to a standard whole blood DNA manual extraction. DNA recovery from whole blood was nearly equivalent to that of the DBCS-based extraction, while DBS-based extraction was 10-fold less sensitive. The detection rate of proviral HIV-1 DNA with DBCS assay was equivalent to whole blood manual extraction (100% concordance), but DBS-extracted samples showed limited concordance (44%). The DBCS assay may prove to be more feasible in resource-limited settings. It may represent a simple and robust point-of-care assay for HIV screening of children, for whom a reference test is still lacking.

Original languageEnglish
Pages (from-to)121-127
Number of pages7
JournalJournal of Virological Methods
Volume170
Issue number1-2
DOIs
Publication statusPublished - Dec 2010

Keywords

  • DBCS (Dried Buffy Coat Spot)
  • DBS (Dried Blood Spot)
  • HIV-1 (human immunodeficiency virus type 1)
  • HIV-1 pol
  • Human β-globin
  • Proviral HIV-1 DNA

ASJC Scopus subject areas

  • Virology

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