Anion-Exchange Filtration and Real-Time PCR for the Detection of a Norovirus Surrogate in Food
2009
Morales-Rayas, Rocío | Wolffs, Petra F.G. | Griffiths, Mansel W.
In the present study, nanoalumina filters were used as a sample preparation step for the concentration of a norovirus surrogate (murine norovirus 1) from food, and this was coupled with a two-step, real-time reverse transcriptase PCR for quantification. The nanoalumina medium was provided in a syringe-filter format, and its binding and elution capacities were tested with different buffers. Among the binding buffers tested (0.1 M Tris-HCl [pH 7.0] with 0.1% Tween 80, 0.1% 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate, or 1 M NaCl), no significant differences were found in the capture capacity of the nanoalumina filters, which was found to be as high as 99.8% of murine norovirus 1 present in the buffer. Elution of 50% of captured viral particles from the filters was possible by using glycine buffer. The desorption capacity of the binding buffers was tested on different inoculated food surfaces. Recoveries of up to 100% from lettuce, raspberries, strawberries, or mussels were obtained with 0.1 M Tris-HCl (pH 7.0) containing 1 M NaCl by using orbital shaking or pipetting. The latter method was more efficient and gave higher recoveries than did orbital shaking. The combination of an efficient desorption-binding-elution buffer with the high concentration capacity of the nanoalumina medium allowed the detection of 10(1) PFU from inoculated produce and 10(5) PFU from inoculated mussels.
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