Development of a novel method for the rapid concentration and detection of norovirus and hepatitis A virus in foods

Noroviruses (NoV) and Hepatitis A viruses (HAV) are the most commonly implicated viruses in foodborne disease. Their transmission is mainly via the fecal-oral route and distribution of contaminated foods has lead to large outbreaks. Thus, it is crucial that contaminated foods be identified promptly. Detection by cell culture is not possible for these viruses so that researchers rely on the reverse transcription polymerase chain reaction (RT-PCR). Prior to detection, however, viruses must be isolated from foods and, as of yet, no one method has been found applicable to a large variety of food matrices. Problems with existing methods include, but are not limited to, the co-extraction of inhibitory molecules and lack of sensitivity. In addition, they are labour-intensive and time consuming. Therefore, the aims of this research project were to develop a novel methodology for the rapid and sensitive isolation and detection of NoV and HAV from select food matrices. In the first part of these studies, TRIzolRTM reagent and the RT-PCR were used to isolate and detect feline calicivirus (FCV) (a norovirus surrogate) from select artificially-inoculated foods. However, the co-extraction of PCR inhibitors resulted in false-negative results. RNA purification methods were then compared prior to detection and Oligo d (T) beads provided for the best sensitivity. However, virus capture was not sufficiently sensitive to be applied to outbreak situations. In the second part of this thesis, the Pathatrix(TM) system was applied to the isolation of HAV and NoV from a variety of foods with the use of positively-charged (cationic) magnetic beads. The use of the Pathatrix(TM) machinery has led to success in detecting a wide range of bacteria from various food matrices, and we were the first to report on its use for the concentration of viruses. When combined with the RT-PCR for detection, the Pathatrix(TM) system was able to detect HAV from inoculated foods at levels typically found in outbreak situations. Though results were not as sensitive with NoV-inoculated foods, the methodology was successful at detecting NoV in outbreak food samples. Furthermore, the cationic beads used in the Pathatrix(TM) system were able to simultaneously isolate two contaminating viruses from food samples. Therefore, the Pathatrix(TM) methodology reported herein is a very sensitive and rapid method that holds promise for the isolation and detection of HAV and NoV in outbreak situations