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

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Abstract
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.