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Journal of Clinical Microbiology, December 2008, p. 3997-4003, Vol. 46, No. 12
0095-1137/08/$08.00+0     doi:10.1128/JCM.00563-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Real-Time PCR with an Internal Control for Detection of All Known Human Adenovirus Serotypes{triangledown}

Marjolein Damen,1,2,3 René Minnaar,1 Patricia Glasius,1 Alwin van der Ham,1 Gerrit Koen,1 Pauline Wertheim,1 and Marcel Beld1*

Laboratory of Clinical Virology, Department of Medical Microbiology, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands,1 Laboratory of Medical Microbiology, OLVG, Amsterdam, The Netherlands,2 Laboratory of the Municipal Health Service, GGD Amsterdam, Amsterdam, The Netherlands3

Received 25 March 2008/ Returned for modification 17 May 2008/ Accepted 7 October 2008

The "gold standard" for the diagnosis of adenovirus (AV) infection is virus culture, which is rather time-consuming. Especially for immunocompromised patients, in whom severe infections with AV have been described, rapid diagnosis is important. Therefore, an internally controlled AV real-time PCR assay detecting all known human AV serotypes was developed. Primers were chosen from the hexon region, which is the most conserved region, and in order to cover all known serotypes, degenerate primers were used. The internal control (IC) DNA contained the same primer binding sites as the AV DNA control but had a shuffled probe region compared to the conserved 24-nucleotide consensus AV hexon probe region (the target). The IC DNA was added to the clinical sample in order to monitor extraction and PCR efficiency. The sensitivity and the linearity of the AV PCR were determined. For testing the specificity of this PCR assay for human AVs, a selection of 51 AV prototype strains and 66 patient samples positive for other DNA viruses were tested. Moreover, a comparison of the AV PCR method described herein with culture and antigen (Ag) detection was performed with a selection of 151 clinical samples. All 51 AV serotypes were detected in the selection of AV prototype strains. Concordant results from culture or Ag detection and PCR were found for 139 (92.1%) of 151 samples. In 12 cases (7.9%), PCR was positive while the culture was negative. In conclusion, a sensitive, internally controlled nonnested AV real-time PCR assay which is able to detect all known AV serotypes with higher sensitivity than a culture or Ag detection method was developed.

* Corresponding author. Present address: KIT Biomedical Research, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands. Phone: 31 20 5665441. Fax: 31 20 6971841. E-mail: m.beld{at}kit.nl

{triangledown} Published ahead of print on 15 October 2008.

Journal of Clinical Microbiology, December 2008, p. 3997-4003, Vol. 46, No. 12
0095-1137/08/$08.00+0     doi:10.1128/JCM.00563-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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