Development of Dual Genotyping Protocol for Agricultural Norovirus Open Access

Abstract

Norovirus is the number one foodborne pathogen globally and is commonly spread through fresh produce. In order to trace back an outbreak, public health officials have to genetically match the strain found in patient stool to a strain found on a food item. Identification involves obtaining the partial sequence of the VP1 gene for the genotype and the RdRp gene for the P-type. Together they comprise the dual genotype of a norovirus strain. The method for dual genotyping stool samples has recently been streamlined such that the VP1 and RdRp sequences can be amplified by a single RT-PCR reaction. Ideally outbreak investigations use uniform methods to identify outbreaks strains across sample types, but food matrices are much more variable in their composition. This represents a gap in methodology that is important to fill given the increasing genetic variability in circulating norovirus strains. Agricultural spaces are important sites of norovirus contamination on produce. I used irrigation water, worker hand rinses, and produce (melon, jalapeño, and tomato) rinses to adapt the single-step stool sample RT-PCR method for use with agricultural samples. In order to overcome issues in low viral load and inhibitory molecules, I went through many rounds of PCR condition optimization, including nested PCR, thermocycler conditions, and reaction solution components. Although using a nested PCR approach showed significant improvement in amplification, it was ultimately shown that the novel primer pair used with stool samples was not fit for widespread use with agricultural samples. While there are lessons learned through the protocol adaptation, these results indicate that alternative methods should be explored for dual genotyping norovirus in agricultural matrices. 

Table of Contents

Introduction............................................................................................................................................... 1

Methods ....................................................................................................................................................13

Results .......................................................................................................................................................21 Discussion.................................................................................................................................................28

References ................................................................................................................................................34 

About this Master's Thesis

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School	Rollins School of Public Health
Department	Environmental Health
Degree	M.P.H.
Submission	Master's Thesis
Language	English
Research Field	Environmental Health
Keyword	genotyping norovirus RT-PCR
Committee Chair / Thesis Advisor	Juan S. Leon, Emory University

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