Citation:

BRINKMAN, N. AND G. FOUT. EVALUATION OF A GENERIC ARRAY APPROACH FOR GENOTYPING NOROVIRUSES. Presented at 106th General Meeting of the American Society of Microbiology, Orlando, FL, May 21 - 25, 2006.

Impact/Purpose:

Overarching Objectives and Links to Multi-Year Planning

This task directly supports the 2003 Drinking Water Research Program Multi-Year Plan's long term goal 2 to "develop new data, innovative tools and improved technologies to support decision making by the Office of Water on the Contaminate Candidate List and other regulatory issues, and implementation of rules by states, local authorities and water utilities" under GRPA Goal 2 (Clean and Safe Water). The overarching objective is to provide the Office of Water, Agency risk assessors and managers, scientists, state regulators, water industry and industry spokes groups with an evaluation of the use of the VFAR approach for determining which human enteric viruses should be on future CCL lists.

Specific Subtask Objectives:

o Conduct a bioinformatics study of human caliciviruses to determine appropriate regions to target for VFAR studies (to be completed by 9/05 in support of Long Term Goal 2 (due 2010)).

o Develop a microarray assay for typing human caliciviruses (to be completed by 9/06 in support of Long Term Goal 2 (due 2010)).

Description:

Noroviruses are the leading cause of nonbacterial gastroenteritis outbreaks in the United States. Because of their potential to contaminate drinking water, the U.S Environmental Protection Agency has included noroviruses on the Contaminant Candidate List (CCL) to assess the public health risk posed by this group of viruses through waterborne routes. Typically, detection and genotype identification of these viruses rely on reverse transcriptase-polymerase chain reaction (RT-PCR) followed by probe hybridization or sequencing of the products. Although sequencing would provide the best way to genotype a sequence, probe hybridization offers the potential for a more rapid and higher throughput analysis of nucleic acids. However, due to the vast genetic diversity of noroviruses, probe hybridization would necessitate the use of multiple probes per genotype. Towards this end, we evaluated the use of a generic array format to examine the feasibility of genotyping noroviruses by probe hybridization. A genomic region of the pol gene is amplified by RT-PCR, and amplicons are used in a single base extension (SBE) reaction where genotype-specific probes are 3-biotinylated. The probes are then hybridized to an Affymetrix GenFlex Tag Array and the resulting fluorescent intensities are calculated. To examine the ability of the SBE-generic array approach to genotype Genogroup I and II norovirus stains, probes that match perfectly, as well as those that contain mismatches of varying number and position were designed. Our results show that, for all strains examined, the perfect-matched tag-probes are labeled in SBE reactions while the mismatched tag-probes are not labeled. Furthermore, in reactions with multiple norovirus templates, only the appropriate perfect-match tag-probes are labeled. These proof-of-concept results demonstrate the utility of this approach when used in combination with additional sequences conducive to genotyping. Moreover, this SBE-generic array format can be adapted to include the simultaneous identification of multiple waterborne pathogens.

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