Flow cytometry detection of infectious rotaviruses in environmental and clinical samples

Abstract

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 Ito(r) 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 x 10(6) and 1/2 x 10(4) 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.

FIG. 1

Rotavirus detection by IIF-FC. Detection of Ito^r P13 virus in MA104 cells (left column) and CaCo-2 cells (right column) is shown. The inoculum used (MPNCU) is indicated in the upper right corner of each plot. Cursors A and B are defined in the text. Plots represent a typical data set.

FIG. 2

Rotavirus RNA detection by a seminested RT-PCR after infection of CaCo-2 cells with different inocula of the Ito^r P13 strain and cell sorting. Lanes: 1, mock-infected cells from cursor A; 2, mock-infected cells from cursor B; 3, mock-infected cells from the rest of the population; 4, cells from cursor A after infection with 0.2 MPNCU; 5, cells from cursor B after infection with 0.2 MPNCU; 6, cells from the rest of the population after infection with 0.2 MPNCU; 7, cells from cursor A after infection with 2 MPNCU; 8, cells from cursor B after infection with 2 MPNCU; 9, cells from the rest of the population after infection with 2 MPNCU; 10, cells from cursor A after infection with 200 MPNCU; 11, cells from cursor B after infection with 200 MPNCU; 12, cells from the rest of the population after infection with 200 MPNCU; M, molecular size marker; −, negative control RT-PCR; +, positive control. Cursors A and B are defined in the text. Numbers on the left and right indicate base pairs.

FIG. 3

Detection of wild-type rotavirus in CaCo-2 cells. The sample (Table 1) is indicated in the upper right corner of each plot. Cursors A and B are defined in the text.