A Custom DNA Tiling Microarray for Detection and Genotyping of Common Foodborne Viruses from Fresh Produce

Introduction: The detection and identification of virus contaminants in food are essential for prevention and investigation of foodborne outbreaks.   In the absence of an efficient cell culture system for most of the common foodborne viruses, molecular methods such as microarray analysis have been applied to detect and genotype these viruses. 

Purpose: The purpose of this study was to assess the effectiveness of a custom DNA tiling microarray for detecting and identifying foodborne viruses from artificially contaminated fresh produce.

Methods: Hepatitis A virus (HAV) strain HM175/18f and norovirus (NoV) strain Minerva2006 were spiked, individually or combined, onto fresh produce (tomato or celery). Viral RNA was either co-extracted with plant RNA or extracted from eluted virus particles. The microarray, which contains overlapping short-oligonucleotide probes covering partial genomes of common foodborne viruses, was custom designed and manufactured by Affymetrix.  Microarray analysis was performed following the modified Affymetrix GeneChip protocol.

Results: We employed two strategies to detect viruses from fresh produce. The first is the total RNA extraction method. Tomato slices were inoculated with HAV, and total RNA was extracted using the published protocol. Poly(A)-positive RNA, including viral RNA, was further purified with Dynabeads Oligo(dT)25 before being applied to microarray. The poly(A) purification was necessary to circumvent the interference of a large quantity of ribosomal RNAs in the total RNA preparations.  The current detection limit is 1E+05 RNA copies. The second method is the elution of virus particles from celery followed by ultracentrifugation. HAV and NoV were inoculated, individually or combined, at a ratio of 1000:1.  Simultaneous detection of two viruses was achievable at as low as 5E+03 copies/virus. 

Significance: We demonstrate the application of a custom DNA tiling microarray for detection of common foodborne viruses from fresh produce.  This method has the potential to address the increasing needs in surveillance and outbreak investigations.