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Applied and Environmental Microbiology, February 2009, p. 618-624, Vol. 75, No. 3
0099-2240/09/$08.00+0     doi:10.1128/AEM.01507-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Detection and Quantification of Noroviruses in Shellfish{triangledown}

Françoise S. Le Guyader,1* Sylvain Parnaudeau,1 Julien Schaeffer,1 Albert Bosch,2 Fabienne Loisy,3 Monique Pommepuy,1 and Robert L. Atmar4

IFREMER, Laboratoire de Microbiologie, Nantes, France,1 Enteric Virus Laboratory, Department of Microbiology, University of Barcelona, Barcelona, Spain,2 CEERAM SAS, La Chapelle sur Erdre, France,3 Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas4

Received 3 July 2008/ Accepted 23 November 2008

Noroviruses (NoVs) are the most common viral agents of acute gastroenteritis in humans, and high concentrations of NoVs are discharged into the environment. As these viruses are very resistant to inactivation, the sanitary consequences are contamination of food, including molluscan shellfish. There are four major problems with NoV detection in shellfish samples: low levels of virus contamination, the difficulty of efficient virus extraction, the presence of interfering substances that inhibit molecular detection, and NoV genetic variability. The aims of this study were to adapt a kit for use with a method previously shown to be efficient for detection of NoV in shellfish and to use a one step real-time reverse transcription-PCR method with addition of an external viral control. Comparisons of the two methods using bioaccumulated oysters showed that the methods reproducibly detected similar levels of virus in oyster samples. Validation studies using naturally contaminated samples also showed that there was a good correlation between the results of the two methods, and the variability was more attributable to the level of sample contamination. Magnetic silica very efficiently eliminated inhibitors, and use of extraction and amplification controls increased quality assurance. These controls increased the confidence in estimates of NoV concentrations in shellfish samples and strongly supported the conclusion that the results of the method described here reflected the levels of virus contamination in oysters. This approach is important for food safety and is under evaluationfor European regulation.

* Corresponding author. Mailing address: Laboratoire de Microbiologie, IFREMER, BP 21105, 44311 Nantes cedex 03, France. Phone: 33 2 40 37 40 52. Fax: 33 2 40 37 40 73. E-mail: sleguyad{at}ifremer.fr

{triangledown} Published ahead of print on 1 December 2008.

Applied and Environmental Microbiology, February 2009, p. 618-624, Vol. 75, No. 3
0099-2240/09/$08.00+0     doi:10.1128/AEM.01507-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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