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 (from3 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.
F. Bonvicini, M. Mirasoli, E. Manaresi, G. A. Gentilomi, A. Roda, G. Gallinella (2013). Development of chemiluminescent assays for the quantitative detection and imaging of 5-bromo-2'deoxyuridine-labeled DNA in parvovirus B19-infected cells. ANALYTICAL AND BIOANALYTICAL CHEMISTRY, 405, 941-949 [10.1007/s00216-012-6372-1].
Development of chemiluminescent assays for the quantitative detection and imaging of 5-bromo-2'deoxyuridine-labeled DNA in parvovirus B19-infected cells
BONVICINI, FRANCESCA;MIRASOLI, MARA;MANARESI, ELISABETTA;GENTILOMI, GIOVANNA ANGELA;RODA, ALDO;GALLINELLA, GIORGIO
2013
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 (from3 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
