Flow cytometry and RT-PCR for rotavirus detection in artificially seeded oyster meat

A flow cytometry (FC)-based method was developed for the detection of rotav irus in oyster meat using simian rotavirus SA11 as a model. To study virus recovery, oyster meat was injected with rotavirus and the oyster extract us ed to infect MA104 cell monolayers. Following varying periods of infection, the cells were recovered and reacted with the monoclonal antibody M60 whic h is specific for the rotavirus group A serotypes 1-4 outer capsid protein, VP7, followed by a second antibody (anti mouse IgG-FITC). A FACScan(R) FC was used to estimate the number of infected cells as well as the level of i nfection. To evaluate the sensitivity of the method, non-inoculated oysters were processed following the same extraction protocol and, at the end, the y were seeded with the same amount of virus used for oyster inoculation. Th is seeded oyster extract was then used to infect MA104 cells and the number of infected cells determined using the same FC procedure. A semi-nested tw o-step PCR for detection of rotavirus nucleic acid was undertaken to compar e the sensitivity of FC with RT-PCR. Using FC, as little as 0.02 flow cytom etry units (fcu) (number of infected cells counted by FC) could be detected after 72 h of cell infection. This is a very similar limit of sensitivity to that obtained with RT-PCR. Both methods are approximately 100 times more sensitive than the plaque-forming units (pfu) assay. (C) 1999 Elsevier Sci ence B.V. All rights reserved.