Calibrated real-time PCR assay for quantitation of human herpesvirus 8 DNA in biological fluids

Francesco Broccolo, Giuseppe Locatelli, Loredana Sarmati, Sara Piergiovanni, Fabrizio Veglia, Massimo Andreoni, Stefano Buttò, Barbara Ensoli, Paolo Lusso, Mauro S. Malnati

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Accurate laboratory tests for the diagnosis of active human herpesvirus 8 (HHV-8) infection are becoming essential to study the pathogenesis of HHV-8-associated tumors and for the clinical management of HHV-8-infected individuals. We have developed a highly sensitive, calibrated quantitative real-time PCR assay for the measurement of cell-free HHV-8 DNA in body fluids, based on the addition of a synthetic DNA calibrator prior to DNA extraction. The calibrator controls each sample for the presence of PCR inhibitors, determines a cutoff value of sensitivity for negative samples, and normalizes positive samples for the efficiency of DNA recovery. The assay shows a wide dynamic range of detection (between 1 and 106 viral genome equivalents/reaction) and a high degree of accuracy even in the presence of high amounts (up to 1 μg) of human genomic DNA. Moreover, the assay has a very high sensitivity (lower detection limit, 10 genome equivalents/ml) and a high degree of reproducibility and repeatability with a coefficient of variation (CV) of

Original languageEnglish
Pages (from-to)4652-4658
Number of pages7
JournalJournal of Clinical Microbiology
Issue number12
Publication statusPublished - Dec 1 2002

ASJC Scopus subject areas

  • Microbiology (medical)
  • Microbiology


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