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Applied and Environmental Microbiology, July 2005, p. 3734-3740, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3734-3740.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Isolation and Detection of Enterovirus RNA from Large-Volume Water Samples by Using the NucliSens miniMAG System and Real-Time Nucleic Acid Sequence-Based Amplification

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

National Institute for Public Health and the Environment (RIVM), Microbiological Laboratory for Health Protection (MGB), Health-Related Water Microbiology, Antonie van Leeuwenhoeklaan 9, 3720 BA Bilthoven, The Netherlands

Received 9 September 2004/ Accepted 10 February 2005

Concentration of water samples is a prerequisite for the detection of the low virus levels that are present in water and may present a public health hazard. The aim of this study was to develop a rapid, standardized molecular method for the detection of enteroviruses in large-volume surface water samples, using a concentration method suitable for the detection of infectious viruses as well as virus RNA. Concentration of water was achieved by a conventional filter adsorption-elution method and ultrafiltration, resulting in a 10,000-fold concentration of the sample. Isolation of virus RNA by a silica-based RNA extraction method was compared with the nonmagnetic and magnetic NucliSens RNA isolation methods. By using the silica-based RNA extraction method in two out of five samples, enterovirus RNA was detected, whereas four out of five samples were positive following RNA isolation with magnetic silica beads. Moreover, estimated RNA levels increased at least 100 to 500 times. Furthermore, we compared enterovirus detection by an in-house reverse transcription (RT)-PCR with a novel commercially available real-time nucleic acid sequence-based amplification (NASBA) assay. We found that the rapid real-time NASBA assay was slightly less sensitive than our in-house RT-PCR. The advantages, however, of a commercial real-time NASBA assay, like the presence of an internal control RNA, standardization, and enormous decrease in turnaround time, makes it an attractive alternative to RT-PCR.

* Corresponding author. Mailing address: National Institute for Public Health and the Environment (RIVM), Microbiological Laboratory for Health Protection (MGB), Health-Related Water Microbiology, 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, July 2005, p. 3734-3740, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3734-3740.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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