FLOW-CYTOMETRY DETECTION OF INFECTIOUS ROTAVIRUSES IN ENVIRONMENTAL AND CLINICAL-SAMPLES

A method for the detection of infectious human rotaviruses based on in fection of CaCo-2 cells and detection of infected cells by indirect im munofluorescence and flow cytometry (IIF-FC) has been developed. The t echnique was validated by performing a seminested reverse transcriptio n-PCR assay with sorted cell populations. The efficiency of the proced ure has been compared with that of the standard method of infection of MA104 cells and ulterior detection by IIF and optical microscopy (IIF -OM) and with that of infection of MA104 cells and detection by IIF-FC , The limit of sensitivity for the detection of the cell-adapted strai n Ito(r) P13, expressed as the most probable number of cytopathogenic units, was established as 200 and 2 for MA104 and CaCo-2 cells, respec tively, by the IIF-FC method. The ratio of infectious virus particles to total virus particles for a wild-type rotavirus was determined to b e 1/2 x 10(6) and 1/2 x 10(4) for IIF-OM with MA104 cells and IIF-FC w ith CaCo-2 cells, respectively. The use of IIF-FC with CaCo-2 cells wa s tested with fecal and water samples and proved to be more effective than the standard procedure for rotavirus detection.