Citation:

BRINKMAN, N. DEVELOPMENT AND EVALUATION OF A MICROARRAY APPROACH TO DETECT AND GENOTYPE NOROVIRUSES IN WATER. Presented at The U.S. EPA Workshop on Innovative Approach to Detecting Microorganisms in Water, Cincinnati, OH, June 18 - 20, 2007.

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, some of which are caused by the ingestion of contaminated water. These viruses are usually detected and genotyped using reverse transcription-polymerase chain reaction (RT-PCR) based methods followed by sequencing. Unfortunately, the accurate detection of noroviruses in environmental samples is often hindered by the co-amplification of non-specific DNA, which can result in the need for further purification of PCR products before accurate sequence information can be obtained. As an alternative to direct sequencing, a generic microarray was evaluated for its ability to genotype norovirus RT-PCR products by probe hybridization. With this approach, RT-PCR amplicons were first mixed with a range of genotype specific probes and single base extension (SBE) reactions were run. This resulted in the labeling of those probes that have sequences complementary to specific RT-PCR products. These genotype-specific probes were then hybridized to an Affymetrix GenFlex Tag Array for detection. Using a standardized, multiplex SBE reaction, the genotyping of representative strains was accomplished and resulted in the generation of specific hybridization patterns, or fingerprints, on the microarray that were diagnostic for the genotype of norovirus detected. Furthermore, the SBE-GenFlex array method was shown to be successful in the genotype identification of noroviruses seeded into tap and Ohio River water samples. This study demonstrates the utility of using a microarray to genotype noroviruses in complex environmental matrices.

Record Details: