Two sample preparation methods based on electrostatic binding were tested to simultaneously separate different viral particles from different food surfaces (lettuce, strawberry, raspberries and green onions). Both methods were evaluated using a multiplex real-time PCR assay designed for detection of hepatitis A virus and norovirus GI and GII. Single and multiplex detection limits were determined as 10(1) viral particles for HAV and norovirus GII, and 10(2) viral particles for norovirus CI using artificial templates, one HAV strain and different norovirus isolates. Manual extraction based on silica columns was found more suitable for viral RNA preparation than an automatic extraction technique. Consistent detection of infectious amounts (2-20 viral particles/g) of HAV and norovirus in different food samples was achievable when the viruses were concentrated using cationically charged filters rather than with cationically charged beads in a flow-through system. Consequently, the developed multiplex detection protocol provides a promising alternative for rapid and simultaneous detection of viral pathogens in foods.
- Real-time PCR