Evaluation of FDA-EVIR Microarray for Detection of Hepatitis A Virus and Norovirus in Inoculated Tomatoes, Green Onions, and Celery.

Foodborne viral contamination of fresh produce has been associated with numerous outbreaks. Detection of such contaminated foods is important for protecting public health. Here, we demonstrate for the first time the capability of the U.S. Food and Drug Administration Enteric Viruses tiling microarray (FDA-EVIR) for rapid molecular identification of hepatitis A virus (HAV) and human norovirus (NoV) extracted from artificially inoculated fresh produce. Two published viral extraction strategies, total RNA extraction or virus particle isolation, were employed to prepare the viral targets. Total RNA extraction method was used on material eluted from tomatoes, using an alkaline Tris-glycine-beef extract (TGBE) buffer. Optimization procedures including DNase treatment and poly(A)-RNA enrichment were adopted for improving microarray sensitivity. For green onions and celery, material was eluted using either Glycine Buffer or TGBE buffer supplemented with pectinase, respectively, then virus particles were concentrated by ultracentrifugation. We also assessed the amount of viral RNA extracted from celery by three commercially-available kits and how well that RNA performed on the FDA-EVIR. Our results confirm that FDA-EVIR can identify common enteric viruses isolated from fresh produce when present as either a single or mixed species of viruses. Using total RNA extraction from tomatoes yielded the limit of detection of 1.0 x 105 HAV genome equivalents (ge) per array input. The limit of detection for viral RNA obtained using ultracentrifugation was 1.2 x 105 HAV ge from green onions and 1.0 x 103 NoV ge from celery per array input. Extending microarray methods to other food matrices should provide important support to surveillance and outbreak investigations.