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Clinical Chemistry 51: 1365-1373, 2005. First published June 10, 2005; 10.1373/clinchem.2004.045088
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Right arrow Molecular Diagnostics and Genetics
(Clinical Chemistry. 2005;51:1365-1373.)
© 2005 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Development of a PCR-Based Assay for Detection, Quantification, and Genotyping of Human Adenoviruses

Barbara Chmielewicz1,a, Andreas Nitsche2, Brunhilde Schweiger3 and Heinz Ellerbrok2

Robert Koch-Institut,1 Projektgruppe, Neuartige Viren’, 2 Zentrum für Biologische Sicherheit 1, and 3 FG12 ‘Virale Infektionen’, Berlin, Germany.

aAddress correspondence to this author at: Robert Koch-Institut, Projektgruppe P11, Nordufer 20, 13353 Berlin, Germany. Fax 49-30-4547-2605; e-mail chmielewiczb{at}rki.de.

Background: Adenoviruses (AdVs) can cause serious disease in immunosuppressed patients, particularly those undergoing allogeneic stem cell transplantation. A method for virus quantification in clinical specimens is essential for monitoring patient adenoviral loads and evaluating new therapeutic approaches.

Methods: We developed a PCR-based assay that combines detection and genotyping of human AdVs, targeting a highly conserved region of the adenoviral genome coding for the DNA polymerase (AdV DPol PCR). We tested the diagnostic applicability of this PCR-based assay by analyzing 159 clinical specimens from children with respiratory disease and comparing the results with those obtained by nested PCR analysis.

Results: The PCR assay detected all currently known AdV serotypes, with a detection limit of ~10 genome equivalents per reaction for 49 of 51 serotypes. No cross-reactivity to human DNA or other DNA viruses was observed. In addition, genotyping of PCR-positive samples was achieved within minutes by fluorescence curve melting analysis in a LightCycler instrument using 6 pairs of hybridization probes, each specific for a single AdV species. Results for clinical specimens were in good concordance with those obtained by nested PCR.

Conclusion: The presented assay is a suitable tool for the detection and genotyping of human AdVs in clinical samples.






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Copyright © 2005 by the American Association for Clinical Chemistry.