Development of a PCR-based assay for detection, quantification, and genotyping of human adenoviruses.
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 approximately 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.


CONCLUSIONS
The presented assay is a suitable tool for the detection and genotyping of human AdVs in clinical samples.
Chmielewicz
Barbara
B
Robert Koch-Institut, Zentrum für Biologische Sicherheit 1, FG12 'Virale Infektionen', Berlin, Germany. chmielewiczb@rki.de
Nitsche
Andreas
A
Schweiger
Brunhilde
B
Ellerbrok
Heinz
H
D000260:Adenoviruses, Human / Q000235:genetics* / Q000302:isolation & purification; D002675:Child, Preschool; D017931:DNA Primers; D015342:DNA Probes; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D005453:Fluorescence; D005838:Genotype; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D043264:Phosphodiesterase I; D016133:Polymerase Chain Reaction; D012120:Respiration Disorders / Q000821:virology
D017931:DNA Primers; D015342:DNA Probes; D004259:DNA-Directed DNA Polymerase; D043264:Phosphodiesterase I