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United States Department of Agriculture

Agricultural Research Service

Research Project: Molecular Biology of Human Pathogens Associated with Food

Location: Produce Safety and Microbiology Research

Title: Low-density microarray technologies for rapid human norovirus genotyping

Authors
item Quiñones, Beatriz
item Lee, Bertram

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: February 21, 2014
Publication Date: N/A

Technical Abstract: Human noroviruses cause up to 21 million cases of foodborne disease in the United States annually and are the most common cause of acute gastroenteritis in industrialized countries. To reduce the burden of foodborne disease associated with viruses, the use of low density DNA microarrays in conjunction with photopolymerization, a rapid and simple colorimetric method, was developed to simultaneously genotype multiple norovirus strains, associated with foodborne disease. A low-density DNA microarray was designed to target the relatively conserved region B at the 3’end of ORF1 encoding RNA-dependent RNA polymerase and also to target the variable region C at the 5’end of ORF2, encoding the major capsid in both norovirus genogroup I and genogroup II, the two genogroups that are responsible for most human disease. In a more recent version of the microarray, additional capture probes were designed to target the variable region D corresponding to the 3’end of ORF2encoding for the capsid in genogroup II. The results obtained after validating the specificity of the array probes indicated that this colorimetric microarray method accurately genotyped the norovirus genogroup I and II strains, resulting in high signal to noise ratio values. To assess the sensitivity for detecting noroviruses with this colorimetric microarray method, various amounts of in vitro RNA transcript specific for a particular strain were tested on the array. The results indicated that the use of photopolymerization with microarrays has a potential detection limit of <10 transcript copies for GI strain and <100-1000 GII transcript copies GII strains. Additional experiments indicated that increasing the amounts of biotin in the target allowed us to improve our detection limit to <100 transcript copies per reaction. Our findings led us to conclude that the use of photopolymerization with low density microarrays enabled the accurate and rapid detection of norovirus genogroup I and II strains.

Last Modified: 10/21/2014
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