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United States Department of Agriculture

Agricultural Research Service

Research Project: PATHOGEN DETECTION AND INTERVENTION METHODS FOR SHELLFISH

Location: Food Safety and Intervention Technologies

Title: Sensitive detection of multiple hepatitis A virus genotypes with a single polony-based assay

Authors
item Chen, Haifeng -
item Richards, Gary
item Yan, Huijun -
item Yan, Xianghe
item Elkins, Chris -

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 4, 2011
Publication Date: N/A

Technical Abstract: Hepatitis A virus (HAV) is one of the major causes of non-bacterial gastroenteritis in humans worldwide. HAV is mostly transmitted via direct person-to-person contact, or by consumption of contaminated foods and water. Since only a few viral particles may cause disease, detection of low levels of HAV in contaminated samples is complex. Here, we describe a single-molecule-based technology, polony, for simultaneous detection and genotyping of HAV. Polony technology amplifies multiple individual HAV viral genomes clonally within a thin acrylamide gel matrix which is attached to a microscope slide. After polony amplification, we genotyped these HAV genomes by performing single base extensions with fluorescent dye-labeled nucleotides. The sensitivity of the polony assay was determined with 10-fold serial dilutions of viral RNA transcript, and its detection sensitivity was approximately 10 viral genomes. For proof of principal, we co-amplified different HAV viral genomes representing subgenotypes IA, IB and IIIA in a single reaction, and correctly determined their genotypes simultaneously based on specific fluorescence features. In addition, we applied this technology to test three other HAV strains. The genotypic result was confirmed by direct DNA sequencing and subsequent phylogenetic analysis. No cross-reactivity was observed with other enteric viruses. The results indicate that this polony method may serve as a valuable tool for the rapid detection and identification of HAV.

Last Modified: 7/23/2014
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