title: Development of a quantitative immunocapture real-time PCR assay for detecting structurally intact adenoviral particles in water.
abstract: Development of rapid, sensitive and specific methods for detection of infectious enteric viruses in water is challenging but crucial for gaining reliable information for risk assessment. An immunocapture real-time PCR (IC-qPCR) was designed to detect jointly the two major viral particle components, i.e. the capsid protein and the viral genome. Targeting both constituents helps circumventing the technical limits of cell culture approaches and the inability of PCR based methods to predict the infectious status. Two waterborne pathogenic virus models, human adenovirus types 2 and 41, were chosen for this study. IC-qPCR showed a detection limit of 10MPNCU/reaction with a dynamic range from 10(2) to 10(6)MPNCU/reaction. Sensitivity was thus 100-fold higher compared to ELISA-based capture employing the same anti-hexon antibodies. After optimisation, application on environmental water samples was validated, and specificity towards the targeted virus types was obtained through the qPCR step. Heat-treated pure samples as well as surface water samples brought evidence that this method achieves detection of encapsidated viral genomes while excluding free viral genome amplification. As a consequence, adenovirus concentrations estimated by IC-qPCR were below those calculated by direct qPCR. The results demonstrate that the IC-qPCR method is a sensitive and rapid tool for detecting, in a single-tube assay, structurally intact and thus potentially infectious viral particles in environmental samples.
authors.affiliation: Centre de Recherche Public-Gabriel Lippmann, Department of Environment and Agro-biotechnologies (EVA), 41 rue du Brill, L-4422 Belvaux, Luxembourg. Electronic address: ogorzaly@lippmann.lu.
authors.forename: Leslie
authors.initials: L
authors.lastname: Ogorzaly
authors.forename: Sébastien
authors.initials: S
authors.lastname: Bonot
authors.forename: Benaissa El
authors.initials: BE
authors.lastname: Moualij
authors.forename: Willy
authors.initials: W
authors.lastname: Zorzi
authors.forename: Henry-Michel
authors.initials: HM
authors.lastname: Cauchie
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D036022:Capsid Proteins / Q000276:immunology; D004279:DNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*; D013997:Time Factors; D014773:Virology / Q000379:methods*; D014871:Water Microbiology
chemical_list: D036022:Capsid Proteins; D004279:DNA, Viral
keywords: Human adenoviruses; Immuncapture real-time PCR; River water; Viral integrity