[en] 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 10 MPNCU/reaction with a dynamic range from 102 to 106 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.
Cauchie, Henry-Michel ; Université de Liège - ULiège > DER Sc. et gest. de l'environnement (Arlon Campus Environ.) > DER Sc. et gest. de l'environnement (Arlon Campus Environ.)
Language :
English
Title :
Development of a quantitative immunocapture real-time PCR assay for detecting structurally intact adenoviral particles in water
Publication date :
December 2013
Journal title :
Journal of Virological Methods
ISSN :
0166-0934
eISSN :
1879-0984
Publisher :
Elsevier Science, Amsterdam, Netherlands
Volume :
194
Pages :
235–241
Peer reviewed :
Peer Reviewed verified by ORBi
Name of the research project :
framework of the PATHOS project (contract number C08/SR/08)
Funders :
framework of the PATHOS project (contract number C08/SR/08), supported by the National Research Fund, Luxembourg
Abd El Galil K.H., El Sokkary M.A., Kheira S.M., Salazar A.M., Yates M.V., Chen W., Mulchandani A. Combined immunomagnetic separation-molecular beacon-reverse transcription-PCR assay for detection of Hepatitis A virus from environmental samples. Appl. Environ. Microbiol. 2004, 70:4371-4374.
Baxter C., Hofmann R., Templeton M., Brown M., Andrews R. Inactivation of adenovirus types 2, 5, and 41 in drinking water by UV light, free chlorine, and monochloramine. J. Environ. Eng. 2007, 133:95-103.
Bofill-Mas S., Albinana-Gimenez N., Clemente-Casares P., Hundesa A., Rodriguez-Manzano J., Allard A., Calvo M., Girones R. Quantification and stability of human adenoviruses and polyomavirus JCPyV in wastewater matrices. Appl. Environ. Microbiol. 2006, 72:7894-7896.
Cromeans T.L., Kahler A.M., Hill V.R. Inactivation of adenoviruses, enteroviruses, and murine norovirus in water by free chlorine and monochloramine. Appl. Environ. Microbiol. 2010, 76:1028-1033.
Deng M.Y., Day S.P., Cliver D.O. Detection of hepatitis A virus in environmental samples by antigen-capture PCR. Appl. Environ. Microbiol. 1994, 60:1927-1933.
Enriquez C.E., Hurst C.J., Gerba C.P. Survival of the enteric adenoviruses 40 and 41 in tap, sea, and waste water. Water Res. 1995, 29:2548-2553.
Haramoto E., Kitajima M., Katayama H., Ohgaki S. Real-time PCR detection of adenoviruses, polyomaviruses, and torque teno viruses in river water in Japan. Water Res. 2010, 44:1747-1752.
Harrach B., Benkö M., Both G., Brown M., Davison A., Echavarria M., Hess M., Jones M., Kajon A., Lehmkuhl H., Mautner V., Mittal S., Wadell G. Family - Adenoviridae. Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses 2012, 125-141. Elsevier, San Diego. A. King, M. Adams, E. Carstens, E. Lefkowitz (Eds.).
Hernroth B.E., Conden-Hansson A.C., Rehnstam-Holm A.S., Girones R., Allard A.K. Environmental factors influencing human viral pathogens and their potential indicator organisms in the blue mussel, Mytilus edulis: the first Scandinavian report. Appl. Environ. Microbiol. 2002, 68:4523-4533.
Hundesa A., Maluquer de Motes C., Albinana-Gimenez N., Rodriguez-Manzano J., Bofill-Mas S., Suñen E., Girones R. Development of a qPCR assay for the quantification of porcine adenoviruses as an MST tool for swine fecal contamination in the environment. J. Virol. Methods 2009, 158:130-135.
Hwang Y.C., Leong O.M., Chen W., Yates M.V. Comparison of a reporter assay and immunomagnetic separation real-time reverse transcription-PCR for the detection of enteroviruses in seeded environmental water samples. Appl. Environ. Microbiol. 2007, 73:2338-2340.
Jothikumar N., Cliver D.O., Mariam T.W. Immunomagnetic capture PCR for rapid concentration and detection of hepatitis A virus from environmental samples. Appl. Environ. Microbiol. 1998, 64:504-508.
Knight A., Li D., Uyttendaele M., Jaykus L.A. A critical review of methods for detecting human noroviruses and predicting their infectivity. Cri. Rev. Microbiol. 2013, 39:295-309.
Le Provost G., Iskra-Caruana M.L., Acina I., Teycheney P.Y. Improved detection of episomal Banana streak viruses by multiplex immunocapture PCR. J. Virol. Methods 2006, 137:7-13.
Lee C.H., Kim S.J. Molecular detection of human enteric viruses in urban rivers in Korea. J. Microbiol. Biotechnol. 2008, 18:1156-1163.
Ling K.S., Wechter W.P., Jordan R. Development of a one-step immunocapture real-time TaqMan RT-PCR assay for the broad spectrum detection of Pepino mosaic virus. J. Virol. Methods 2007, 144:65-72.
Myrmel M., Rimstad E., Wasteson Y. Immunomagnetic separation of a Norwalk-like virus (genogroup I) in artificially contaminated environmental water samples. Int. J. Food Microbiol. 2000, 62:17-26.
Nuanualsuwan S., Cliver D.O. Capsid functions of inactivated human picornaviruses and feline calicivirus. Appl. Environ. Microbiol. 2003, 69:350-357.
Ogorzaly L., Bertrand I., Paris M., Maul A., Gantzer C. Occurrence, survival, and persistence of human adenoviruses and F-specific RNA phages in raw groundwater. Appl. Environ. Microbiol. 2010, 76:8019-8025.
Park Y.B., Cho Y.H., Jee Y.M., Ko G. Immunomagnetic separation combined with real-time reverse transcriptase PCR assays for detection of norovirus in contaminated food. Appl. Environ. Microbiol. 2008, 74:4226-4230.
Peng X., Luo W., Zhang J., Wang S., Lin S. Rapid detection of Shigella species in environmental sewage by an immunocapture PCR with universal primers. Appl. Environ. Microbiol. 2002, 68:2580-2583.
Peroni L.A., Reis J.R.R., Coletta-Filho H.D., de Souza A.A., Machado M.A., Stach-Machado D.R. Assessment of the diagnostic potential of Immmunocapture-PCR and Immuno-PCR for Citrus Variegated Chlorosis. J. Microbiol. Methods 2008, 75:302-307.
Price E.P., Dale J.L., Cook J.M., Sarovich D.S., Seymour M.L., Ginther J.L., Kaufman E.L., Beckstrom-Sternberg S.M., Mayo M., Kaestli M., Glass M.B., Gee J.E., Wuthiekanun V., Warner J.M., Baker A., Foster J.T., Tan P., Tuanyok A., Limmathurotsakul D., Peacock S.J., Currie B.J., Wagner D.M., Keim P., Pearson T. Development and validation of Burkholderia pseudomallei-specific real-time PCR assays for clinical, environmental or forensic detection applications. PLoS ONE 2012, 7:e37723.
Rimhanen-Finne R., Hörman A., Ronkainen P., Hänninen M.L. An IC-PCR method for detection of Cryptosporidium and Giardia in natural surface waters in Finland. J. Microbiol. Methods 2002, 50:299-303.
Rodriguez-Lazaro D., Cook N., Ruggeri F.M., Sellwood J., Nasser A., Nascimento M.S.J., D'Agostino M., Santos R., Saiz J.C., Rzetutka A., Bosch A., Girones R., Carducci A., Muscillo M., Kovac K., Diez-Valcarce M., Vantarakis A., von Bonsdorff C.H., de Roda Husman A.M., Hernandez M., van der Poel W.H.M. Virus hazards from food, water and other contaminated environments. FEMS Microbiol. Rev. 2012, 36:786-814.
Sharman M., Thomas J.E., Dietzgen R.G. Development of a multiplex immunocapture PCR with colourimetric detection for viruses of banana. J. Virol. Methods 2000, 89:75-88.
Sibley S.D., Goldberg T.L., Pedersen J.A. Detection of known and novel adenoviruses in cattle wastes via broad-spectrum primers. Appl. Environ. Microbiol. 2011, 77:5001-5008.
Silva H.D., Garcia-Zapata M.T.A., Anunciaçao C.E. Why the use of adenoviruses as water quality virologic marker?. Food Environ. Virol. 2011, 3:138-140.
Skraber S., Gantzer C., Helmi K., Hoffmann L., Cauchie H.M. Simultaneous concentration of enteric viruses and protozoan parasites: a protocol based on tangential flow filtration and adapted to large volumes of surface and drinking waters. Food. Environ. Virol. 2009, 1:66-76.
Verheyen J., Timmen-Wego M., Laudien R., Boussaad I., Sen S., Koc A., Uesbeck A., Mazou F., Pfister H. Detection of adenoviruses and rotaviruses in drinking water sources used in rural areas of Benin, West Africa. Appl. Environ. Microbiol. 2009, 75:2798-2801.
Wigginton K.R., Pecson B.M., Sigstam T., Bosshard F., Kohn T. Virus inactivation mechanisms: impact of disinfectants on virus function and structural integrity. Environ. Sci. Technol. 2012, 46:12069-12078.
Wong K., Xagoraraki I. Quantitative PCR assays to survey the bovine adenovirus levels in environmental samples. J. Appl. Microbiol. 2010, 109:605-612.
Wu J., Meng C., Shang H., Rong S., Zhang C., Hong J., Zhou X. Monoclonal antibody-based triple antibody sandwich-enzyme-linked immunosorbent assay and immunocapture reverse transcription-polymerase chain reaction for Odontoglossum ringspot virus detection. J. Virol. Methods 2011, 171:40-45.
Wyer M.D., Wyn-Jones A.P., Kay D., Au-Yeung H.K.C., Girones R., Lopez-Pila J., de Roda Husman A.M., Rutjes S., Schneider O. Relationships between human adenoviruses and faecal indicator organisms in European recreational waters. Water Res. 2012, 46:4130-4141.
Wyn-Jones A.P., Carducci A., Cook N., D'Agostino M., Divizia M., Fleischer J., Gantzer C., Gawler A., Girones R., Höller C., de Roda Husman A.M., Kay D., Kozyra I., López-Pila J., Muscillo M., José Nascimento M.S., Papageorgiou G., Rutjes S., Sellwood J., Szewzyk R., Wyer M. Surveillance of adenoviruses and noroviruses in European recreational waters. Water Res. 2011, 45:1025-1038.
Yang W., Gu A.Z., Zeng S.Y., Li D., He M., Shi H.C. Development of a combined immunomagnetic separation and quantitative reverse transcription-PCR assay for sensitive detection of infectious rotavirus in water samples. J. Microbiol. Methods 2011, 84:447-453.
