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P2-126 Evaluation of Molecular Methods for Detection and Genotyping of Norovirus

Tuesday, August 5, 2014
Exhibit Hall D (Indiana Convention Center)
Christine Yu, U.S. Food and Drug Administration, Laurel, MD
Kaoru Hida, U.S. Food and Drug Administration, Laurel, MD
Efi Papafragkou, U.S. Food and Drug Administration, Laurel, MD
Zhihui Yang, U.S. Food and Drug Administration, Laurel, MD
Susan Leonard, U.S. Food and Drug Administration, Laurel, MD
Michael Kulka, U.S. Food and Drug Administration, Laurel, MD
Introduction: The detection and identification of virus contaminants are essential for investigation and prevention of foodborne outbreaks.   Norovirus is one of the most important causative agents of foodborne gastroenteritis.  Due to the lack of a cell culture system, molecular methods have been applied to detect and genotype norovirus. 

Purpose: The purpose of this study was to assess the effectiveness and suitability of the different molecular methods for detecting and identifying foodborne pathogens from food and clinical samples.

Methods: We adopted three approaches, RT-qPCR, DNA tiling microarray, and NextGen sequencing (NGS).  RT-qPCR was evaluated in detection of human norovirus (NoV), together with extraction control, murine norovirus (MNV), following extraction by the ultracentrifugation method (with PVP) from a variety of foods.  Microarray and NGS were applied to detection and genotyping of NoV present in human fecal samples.

Results: While MNV was extracted with a recovery rate ranging from 10% to 40% and detected efficiently by RT-qPCR, none of the foods implicated in the recent noro GII outbreak tested positive for NoV.  DNA microarray can detect and genotype norovirus when the sequence of the target virus is more than an 80% match to the probes, the signal intensity directly correlated to the % match.  The depth of coverage (> 200X) obtained from NGS of the stool sample is sufficient to generate useful viral sequence contigs leading to de novo assembly and match to published GenBank sequences. 

Significance: We demonstrate the applications of real-time RT-qPCR, microarray, and NGS for detection of norovirus in clinical or food samples.  These methods have the potential to address the differential needs in surveillance and outbreak investigations.

Back to: Poster Session - Pathogens, Epidemiology, Novel Laboratory Methods, Food Defense, Communication Outreach and Education, General Microbiology, Dairy and Other Food Commodities, Food Toxicology
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