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Detection of Norovirus and Hepatitis A in Raw Produce using RT-PCR Protocols


Noroviruses (NoV) and hepatitis A viruses (HAV) are the most common viruses associated with foodborne illnesses and are primarily transmitted through the fecal-oral route. High-risk foods include fruits and vegetables since they may be contaminated by irrigation, by washing with fecal-contaminated water or by infected food handlers. Currently, reverse transcriptase PCR (RT-PCR) has been successfully used to detect NoV and HAV in clinical samples. However, detection of the virus in naturally contaminated food such as fresh produce has been more difficult due to low virus numbers. RT-PCR is currently the primary detection method for NoV and HAV; however, real-time RT-PCR is a faster, more sensitive method than conventional RT-PCR. Since the targeted RNA is simultaneously amplified and detected, post-amplification steps such as gel electrophoresis or sequencing necessary to confirm the identity of PCR products are eliminated, saving time, labor and money. Real-time RT-PCR technology can also generate quantitative data, unlike conventional RT-PCR.
Challenges for the development of a sensitive and specific RT-PCR detection method include the elution and concentration of low virus numbers in naturally contaminated foods and the removal of inherent PCR inhibitors. Recently, elution/concentration procedures, extraction procedures and RT-PCR assays for NoV and HAV detection in green onions were developed by scientists at the Canadian Food Inspection Agency (CFIA) and Agriculture and Agrifood Canada (AAFC). In collaboration with these scientists, we propose to extend the validation of these methods by adapting and optimizing these methods for HAV and NoV detection in fresh produce such as cut and uncut cabbage, lettuce and green peppers, which are often eaten raw. The goal of this project is to adapt and optimize a real-time RT- PCR method or develop an alternative method for the optimal detection of the virus. Furthermore, elution/concentration and extraction methods will be adapted to optimize concentration of HAV and NoV and removal of PCR inhibitors. The selected sample preparation procedures will then be combined with the real-time PCR assay and validated using artificially-contaminated samples.

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Expected Impact of Project Outcomes on Food Safety in Ontario:
Noroviruses (NoV) and hepatitis A virus (HAV) are the most common viruses associated with foodborne illnesses. There is a public health concern regarding the rapid spread of these two viruses in sporadic and outbreak situations associated with the consumption of contaminated foods. It is estimated that 67 percent of foodborne illnesses might be caused by NoV in the United States. In Ontario about 50 percent of the HAV reported cases are associated with foodborne or waterborne transmission. The primary transmission mode for NoV and HAV is by the fecal-oral route and these viruses are either transmitted directly by person-to-person contact or indirectly via contaminated food or water. These two viruses can remain stable in the environment for long periods and have been found to be resistant to commercial disinfectants, heat and pH changes
Several major steps are important in the development of a sensitive and specific real-time RT-PCR method for detection of low numbers of NoV and HAV in foods: (1) elution and concentration of virus from the matrix, (2) extraction/purification of viral RNA (3) optimization of the PCR mixture including primers and (4) detection/quantification. In this study we propose to optimize and validate these steps for detection of these viruses in produce, especially for routine testing of food samples submitted in sporadic and outbreak cases. A major benefit from this study is to provide standardized methods for NoV and HAV detection in various diagnostic labs in Ontario and across Canada. It will also be a valuable tool for generating surveillance and risk assessment data on high-risk foods such as fresh produce and also in the elucidation of the sources of viral contamination.
<P> For more information, please visit the <a href="; target="_blank">Ontario Ministry of Agriculture, Food & Rural Affairs (OMAFRA) Food Safety Research Program</a>.

Odumeru, Joseph
University of Guelph
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