Development of chemiluminescent assays for the quantitative detection and imaging of 5-bromo-2deoxyuridine-labeled DNA in parvovirus B19-infected cells

摘要

Incorporation of exogenous analogues is a widely used method to evaluate DNA synthesis in cultured cells exposed to exogenous factors such as infectious agents. Herein, two new quantitative methodologies exploiting ultrasensitive chemiluminescence (CL) detection of 5-bromo-2-deoxyuridine (BrdU) have been developed: a CL microscope imaging assay to evaluate BrdU labelling at single-cell level and a CL dot-blot assay to measure the amounts of DNA produced in the course of an in vitro infection of proliferating cells. The assays have been optimized on UT7/EpoS1 cells cultured in presence of different concentrations of BrdU (from 3 to 100 μM) and used to monitor parvovirus B19 (B19) life cycle in infected cells. The CL microscope imaging assay provided a detailed localization of BrdU-labelled nuclei allowing to count positive cells and measure their related CL intensity signals. The CL dot-blot assay, coupled with a B19 capture procedure performed with a specific peptide nucleic acid probe, has been designed to discriminate and selectively quantify cellular and viral BrdU-labelled genomes. Quantitative evaluation of BrdU-labelled B19 DNA has been achieved by means of a CL calibration curve. The high detectability, down to 2∈×∈10~6 B19 genome copies, and the linear range extending up to 5∈×∈10~8 copies make the method suitable to evaluate the amounts of B19 DNA produced throughout a replicative viral cycle.