title: Development of chemiluminescent assays for the quantitative detection and imaging of 5-bromo-2'deoxyuridine-labeled DNA in parvovirus B19-infected cells.
abstract: 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.
authors.affiliation: Department of Pharmacy and BioTechnology, University of Bologna, Bologna, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Bonvicini
authors.forename: Mara
authors.initials: M
authors.lastname: Mirasoli
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Manaresi
authors.forename: Giovanna Angela
authors.initials: GA
authors.lastname: Gentilomi
authors.forename: Aldo
authors.initials: A
authors.lastname: Roda
authors.forename: Giorgio
authors.initials: G
authors.lastname: Gallinella
mesh_terms: D001973:Bromodeoxyuridine / Q000737:chemistry; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D010322:Parvoviridae Infections / Q000175:diagnosis / Q000821:virology*; D016732:Parvovirus B19, Human / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D004279:DNA, Viral; D001973:Bromodeoxyuridine