| In the present research, immunoseparation and ionic binding based methods were developed to separate pathogens from different food matrices prior to detection by real-time PCR. Flow-through immunomagnetic separation was used to concentrate Campylobacter cells from culture and chicken skin samples. In order to determine the recovery of cells by the beads, different parameters were tested. As resolved by a SYBR Green real-time PCR assay, no more than 1% of the cells captured by the beads were detectable and therefore, different enrichment times were evaluated to increase Campylobacter numbers. Four strains showed that after 8 hours enrichment, it was possible to detect 103 CFU/25 g using immunomagnetic concentration as a pre-PCR step. Ionic binding was evaluated next to improve the recovery of microorganisms present in a sample, and therefore, a new protocol using nanoalumina media was developed for the concentration of viruses from produce. Murine norovirus was used as a surrogate for human norovirus to optimize desorption, binding and elution from cationically charged filters. Different produce and mussels were inoculated and processed using anion exchange filtration. After concentration, the Taqman real-time PCR assay was able to detect as low as 101 PFU/50 g of produce and 105 PFU/10 g of mussels. Once optimization of the protocol was achieved, a multiplex Taqman real-time RT-PCR assay for the detection of norovirus GI and GII and Hepatitis A in a single assay was developed. Manual extraction based on silica columns was more suitable for nucleic acid extraction from Hepatitis A virus and murine norovirus after comparison with automatic extraction. Concentration methods tested were based on ionic binding using both nanoalumina media and cationically charged beads. Real-time RT-PCR revealed that anion exchange filtration provided more consistent viral elution, and therefore detection, than cationically charged beads from different food surfaces. Moreover, higher recoveries were obtained, 12--33% of HAV and 5--16% of norovirus GII. Consequently, filtration through nanoalumina media resulted in a simple and fast alternative for detection of low numbers of diverse viral particles that might be present in food. |
