Purpose: To develop a rapid and simple genotyping tool for foodborne viruses, a focused, low-density DNA microarray was developed in conjunction with a rapid and high-throughput fluorescent method.
Methods: Probes (25-mer) were designed to target the NoV variable genomic region C at the 5’-end of ORF2, encoding the major capsid in both NoV genogroup I (GI) and genogroup II (GII). Additional capture probes were designed to target region 3, the VP1/P2A junction region at the end of the capsid region and beginning of the non-structural proteins in HAV genotypes IA and IB.
Results: Validation experiments indicated that this fluorescent array method accurately genotyped NoV GI and GII reference strains, resulting in high fluorescent signal values. To determine the assay sensitivity threshold, various amounts of in vitro cRNA transcript were tested on the array, and the results indicated that the use of microarrays has a potential detection limit of < 10 transcript copies for NoV GI and GII strains and < 100-1000 GII transcript copies of HAV strains. To determine the feasibility of using this array method for detecting foodborne viruses in environmental samples, water in lakes, streams, rivers and ponds that were part of watersheds adjacent to a leafy vegetable production region in the Salinas Valley along the Central California Coast were sampled. After conducting the genotyping analysis of over 100 water samples, NoV genotypes GI.2, GI.4, and HAV IB were the more commonly identified with a prevalence percentage above 50%. A lower prevalence below 40% was observed for GI.3a, G1.6, GII.1, GII.2, GII.4, GII.6, and GII.12.
Significance: Our findings led us to conclude that the use of low density microarrays in conjunction with a fluorescent signal amplification method enabled the accurate and rapid detection of NoV GI and GII strains as well as HAV from environmental samples.