A Custom DNA Tiling Microarray for Detection and Genotyping of Norovirus from Mixed Foodborne Virus Samples

Introduction: The detection and identification of virus contaminants in food and environmental samples are essential for investigation and prevention of foodborne outbreaks. Norovirus (NoV) is one of the most important causative agents of foodborne gastroenteritis.  Without a cell culture system, molecular methods such as microarray analysis have been applied to detect and genotype norovirus. 

Purpose: The purpose of this study was to assess the effectiveness and reliability of a custom DNA tiling microarray for detecting and identifying norovirus from mixed common foodborne virus samples.

Methods: Hepatitis A virus (HAV) and coxsackievirus (CV) RNAs were extracted from virus-infected cell culture supernatants.  A GII.4 NoV genome representing an exact match for a selected probeset on the array was commercially synthesized and cloned into an expression vector.  NoV RNA transcripts were produced from in vitro transcription.  Tiling Microarray was custom designed and manufactured by Affymetrix.  Microarray analysis was performed following the modified Affymetrix GeneChip protocol.

Results: RNAs from three unrelated viruses, CV-B1, HAV-HM175/18f, and NoV-GII.4, were individually, or as a mixture, reverse transcribed into cDNA and hybridized to the microarray.  Simultaneous detection of all three viruses was achievable at the input level of 10⁴ copies/virus.  Surprisingly, array signal intensities for a given virus was higher as a function of input when detected as a mixture rather than when examined individually.  Importantly, the hybridization signal pattern specific for each virus was highly preserved when obtained as a mixture, and there was no noticeable cross-hybridization among the three viruses.  As a result, successful detection and identification of three differing virus species was attained at lower than previously published input quantities.

Significance: We demonstrate the application of a tiling microarray for detection of norovirus in mixed foodborne virus samples.  This method has the potential to address the ever increasing needs in surveillance and outbreak investigations.