Use of flow cytometry for rapid, quantitative detection of poliovirus-infected cells via TAT peptide-delivered molecular beacons

Abstract

Rapid and efficient detection of viral infection is crucial for the prevention of disease spread during an outbreak and for timely clinical management. In this paper, the utility of Tat peptide-modified molecular beacons (MBs) as a rapid diagnostic tool for the detection of virus-infected cells was demonstrated. The rapid intracellular delivery mediated by the Tat peptide enabled the detection of infected cells within 30 s, reaching saturation in signal in 30 min. This rapid detection scheme was coupled with flow cytometry (FC), resulting in an automated, high-throughput method for the identification of virus-infected cells. Because of the 2-order-of-magnitude difference in fluorescence intensity between infected and uninfected cells, as few as 1% infected cells could be detected. Because of its speed and sensitivity, this approach may be adapted for the practical diagnosis of multiple viral infections.

Fig 1

Real-time detection of PV1 in BGMK cells. Cells were infected with 10 PFU PV1 for 18 h, detached and incubated with 1 μM MB-PV1, and monitored in real time from 0 to 30 min using a fluorescence microscope. The corresponding phase contrast picture is shown.

Fig 2

Quantification of PV1-infected BGMK cells using flow cytometry. Confluent monolayers of BGMK cells (1.5 × 10⁵ cells) in 8-well chamber slides were infected with 10⁶ PFU of PV1. After 18 h of infection, cells were detached from the monolayer by using trypsin and mixed with uninfected cells to represent different percentages of infection (0%, 1%, 2%, 5%, 10%, 20%, and 100%). Cells were washed with 10% FBS followed by 1× TBS with 3 mM EDTA (pH 8.0), mixed with 1 μM MB-PV1, and subjected to flow cytometry. The corresponding fluorescence images for selected samples are included.

Fig 3

The correlation between the percentage of fluorescent cells measured by FC and the percentage of infected cells in the mixture. Data shown are the mean values (± standard deviations) obtained from 3 independent experiments.

Fig 4

Comparison of fluorescent cell detection by FC and fluorescence microscopy. Various percentages of PV1-infected cells (0%, 1%, 2%, 5%, 10%, 20%, and 100%) were incubated with 1 μM MB and analyzed using either flow cytometry or fluorescence microscopy. The fluorescent and differential interference contrast (DIC) images from 5 different fields within the chamber well were captured using ×20 magnification, and the percentage of fluorescent cells was calculated. Data shown are the mean values (± standard deviations) obtained from 3 independent experiments.

Fig 5

Quantification of PV1-infected cells using flow cytometry. Confluent monolayers of BGMK cells were infected with various doses of PV1 from 0 PFU to 10⁵ PFU for 18 h. The percentages of infected cells shown in the graph were determined by counting the number of fluorescent cells and dividing it by the total number of cells counted.