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.
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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.