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Applied and Environmental Microbiology, April 2005, p. 1870-1875, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.1870-1875.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Rapid and Sensitive Detection of Noroviruses by Using TaqMan-Based One-Step Reverse Transcription-PCR Assays and Application to Naturally Contaminated Shellfish Samples

Narayanan Jothikumar,1,2 James A. Lowther,3 Kathleen Henshilwood,3 David N. Lees,3 Vincent R. Hill,2 and Jan Vinjé1*

Environmental Science and Engineering, University of North Carolina, Chapel Hill, North Carolina,1 Coordinating Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia,2 The Centre for Environment Fisheries and Aquaculture Science, Weymouth, England3

Received 7 October 2004/ Accepted 4 November 2004

Noroviruses (NoV), which are members of the family Caliciviridae, are the most important cause of outbreaks of acute gastroenteritis worldwide and are commonly found in shellfish grown in polluted waters. In the present study, we developed broadly reactive one-step TaqMan reverse transcription (RT)-PCR assays for the detection of genogroup I (GI) and GII NoV in fecal samples, as well as shellfish samples. The specificity and sensitivity of all steps of the assays were systematically evaluated, and in the final format, the monoplex assays were validated by using RNA extracted from a panel of 84 stool specimens, which included NoV strains representing 19 different genotypes (7 GI, 11 GII, and 1 GIV strains). The assays were further validated with 38 shellfish cDNA extracts previously tested by nested PCR. Comparison with a recently described real-time assay showed that our assay had significantly higher sensitivity and was at least as sensitive as the nested PCR. For stool specimens, a one-step duplex TaqMan RT-PCR assay performed as well as individual genogroup-specific monoplex assays. All other enteric viruses examined were negative, and no cross-reaction between genogroups was observed. These TaqMan RT-PCR assays provide rapid (less than 90 min), sensitive, and reliable detection of NoV and should prove to be useful for routine monitoring of both clinical and shellfish samples.

* Corresponding author. Mailing address: Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, NC 27599. Phone: (919) 966-7317. Fax: (919) 966-4711. E-mail: janvinje{at}email.unc.edu.

Applied and Environmental Microbiology, April 2005, p. 1870-1875, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.1870-1875.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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