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Appl Environ Microbiol, July 1998, p. 2392-2396, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Flow Cytometry Detection of Infectious Rotaviruses in Environmental and Clinical Samples

F. Xavier Abad, Rosa M. Pintó, and Albert Bosch*

Department of Microbiology, University of Barcelona, 08028 Barcelona, Spain

Received 24 June 1997/Accepted 31 March 1998

A method for the detection of infectious human rotaviruses based on infection of CaCo-2 cells and detection of infected cells by indirect immunofluorescence and flow cytometry (IIF-FC) has been developed. The technique was validated by performing a seminested reverse transcription-PCR assay with sorted cell populations. The efficiency of the procedure 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 strain Itor P13, expressed as the most probable number of cytopathogenic units, was established as 200 and 2 for MA104 and CaCo-2 cells, respectively, by the IIF-FC method. The ratio of infectious virus particles to total virus particles for a wild-type rotavirus was determined to be 1/2 × 106 and 1/2 × 104 for IIF-OM with MA104 cells and IIF-FC with CaCo-2 cells, respectively. The use of IIF-FC with CaCo-2 cells was tested with fecal and water samples and proved to be more effective than the standard procedure for rotavirus detection.

* Corresponding author. Mailing address: Dep. Microbiology, University of Barcelona, School of Biology, Av. Diagonal, 645, 08028 Barcelona, Spain. Phone: (34 93) 402.14.85. Fax: (34 93) 411.05.92. E-mail: albert{at}bio.ub.es.

Appl Environ Microbiol, July 1998, p. 2392-2396, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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