1a.Objectives (from AD-416):
Development and validation of sensitive, rapid and practical methods to detect and genotype human noroviruses in relevent sample matrices (food, water,environmental, clinical).
1b.Approach (from AD-416):
Use DNA microarray platform in conjunction with rapid colorimetric procedure (ampliPHOX system) for genotyping human noroviruses.
To develop a method for genotyping foodborne-associated noroviruses, low-density DNA microarrays were designed to be used in conjunction with a rapid colorimetric method that provides an inexpensive alternative to fluorescence detection by using reagents and instrumentation suitable for routine pathogen surveillance. For constructing a norovirus-specific microarray, 25-mer and 35-mer capture probes were designed to target conserved and variable regions of the norovirus genome to selectively genotype 13 norovirus strains associated with foodborne illness.
Validation experiments indicated that shorter probes (25-mer) allowed a more accurate and specific genotyping of norovirus strains than using longer probes (35-mer). Sensitivity assays demonstrated a detection threshold of 100 copies of cRNA transcripts, corresponding to a similar sensitivity for the detection of respiratory viruses with this colorimetric microarray method. The feasibility of using this colorimetric microarray method for detecting noroviruses in environmental samples is currently being employed in the analysis of water samples from relevant watersheds in the produce production regions in California. The present study has developed a novel, simple and inexpensive genotyping assay for noroviruses, allowing the identification of risk factors to help reduce contamination of noroviruses in pre- and post-harvest environments. This is related to parent project Objective Number 4 aimed at the development of rapid, simple and inexpensive multiplex assays for pathogen detection and virulence characterization using novel technology for use in surveillance and outbreak epidemiology.