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Applied and Environmental Microbiology, August 2006, p. 5349-5358, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.00751-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Real-Time Detection of Noroviruses in Surface Water by Use of a Broadly Reactive Nucleic Acid Sequence-Based Amplification Assay

Saskia A. Rutjes,* Harold H. J. L. van den Berg, Willemijn J. Lodder, and Ana Maria de Roda Husman

National Institute of Public Health and the Environment (RIVM), Centre for Infectious Disease Control (CIb), P.O. Box 1, Antonie van Leeuwenhoeklaan 9, 3720 BA Bilthoven, The Netherlands

Received 31 March 2006/ Accepted 8 June 2006

Noroviruses are the most common agents causing outbreaks of viral gastroenteritis. Outbreaks originating from contaminated drinking water and from recreational waters have been described. Due to a lack of cell culture systems, noroviruses are detected mostly by molecular methods. Molecular detection assays for viruses in water are often repressed by inhibitory factors present in the environment, like humic acids and heavy metals. To study the effect of environmental inhibitors on the performance of nucleic acid sequence-based amplification (NASBA), we developed a real-time norovirus NASBA targeting part of the RNA-dependent RNA polymerase (RdRp) gene. Specificity of the assay was studied with 33 divergent clones that contained part of the targeted RdRp gene of noroviruses from 15 different genogroups. Viral RNA originated from commercial oysters, surface waters, and sewage treatment plants in The Netherlands. Ninety-seven percent of the clones derived from human noroviruses were detected by real-time NASBA. Two clones containing animal noroviruses were not detected by NASBA. We compared the norovirus detection by real-time NASBA with that by conventional reverse transcriptase PCR (RT-PCR) with large-volume river water samples and found that inhibitory factors of RT-PCR had little or no effect on the performance of the norovirus NASBA. This consequently resulted in a higher sensitivity of the NASBA assay than of the RT-PCR. We show that by combining an efficient RNA extraction method with real-time NASBA the sensitivity of norovirus detection in water samples increased at least 100 times, which consequently has implications for the outcome of the infectious risk assessment.

* Corresponding author. Mailing address: National Institute of Public Health and the Environment (RIVM), Centre for Infectious Disease Control (CIb), P.O. Box 1, Antonie van Leeuwenhoeklaan 9, 3720 BA Bilthoven, The Netherlands. Phone: 31 30 274 3272. Fax: 31 30 274 4434. E-mail: saskia.rutjes{at}rivm.nl.

Applied and Environmental Microbiology, August 2006, p. 5349-5358, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.00751-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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