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. 2004 Oct;31(2):116-22.
doi: 10.1016/j.jcv.2004.04.003.

Development of a PCR-and hybridization-based assay (PCR Adenovirus Consensus) for the detection and the species identification of adenoviruses in respiratory specimens

Astrid Vabret  1 , Stéphanie Gouarin, Martine Joannes, Come Barranger, Joëlle Petitjean, Sandrine Corbet, Jacques Brouard, Françoise Lafay, Jean-François Duhamel, Bernard Guillois, François Freymuth
Affiliations

Development of a PCR-and hybridization-based assay (PCR Adenovirus Consensus) for the detection and the species identification of adenoviruses in respiratory specimens

Astrid Vabret et al. J Clin Virol. 2004 Oct.

Abstract

Background: Antigen detection assays and viral isolation techniques are routinely used to detect adenoviruses (Ad) associated with respiratory infections, and the value of the polymerase chain reaction (PCR) has recently been assessed.

Objectives: This paper describes a PCR-hybridization-immunoenzymatic assay (PCR Adenovirus consensus) used to detect Ad and identify Ad species in respiratory specimens.

Results: On seven representative serotypes Ad 12, Ad 3, Ad 7, Ad 11, Ad 1, Ad 8, Ad 4, the mean genome equivalents per ml and the mean 50% infectious doses per ml were 10(6.3)and 10(4), respectively. Using 362 nasal aspirates from children, Ad were detected by immunofluorescence (IF) and culture in 97 cases (27%), by the PCR-Ad hexon method in 107 cases (29.5%) and by the PCR Adenovirus Consensus method in 113 cases (31.2%); 13 samples were found positive by both PCR and negative by the IF and culture methods; five samples were only positive according to the PCR Adenovirus Consensus) method. The sensitivity, specificity, predictive positive value and predictive negative value of the PCR Adenovirus Consensus method were 97.9%, 93.2%, 84%, 99.1%, respectively. The method identified the species (sp) from 91 positive amplicons: 1 Ad sp A, 44 Ad sp B, 42 Ad sp C, 3 Ad sp E, and 1 Ad sp F; 85 isolates were identified by IF or the neutralisation in culture, and 86 by a PCR-RE digestion method. The PCR Adenovirus Consensus detected six positive samples that were negative according to the IF and culture methods, and it identified the precise species of nine IF-positive and culture-negative nasal aspirates.

Conclusion: The PCR Adenovirus Consensus technique is more efficient than the classical IF or culture techniques for the detection of Ad in respiratory samples. An internal control is included to validate the screening results, and specific probes are used to identify the Ad species.

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Figures

Fig. 1
Fig. 1
Ethidium bromide-stained agarose gel showing VA RNA gene region PCR products derived from Ad representative of the indicated species. Bands of two size ranges were observed: 290–340 bp for one gene VA RNA Ad 12, Ad 11, Ad 40; and 460–560 bp for two genes VA RNA Ad 7, Ad 5, Ad 8, Ad 4. Lane M: 100 bp molecular weight marker
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