Development of chemiluminescent assays for the quantitative detection and imaging of 5-bromo-2 %26apos; deoxyuridine-labeled DNA in parvovirus B19-infected cells

作者：Bonvicini Francesca; Mirasoli Mara^*; Manaresi Elisabetta; Gentilomi Giovanna Angela; Roda Aldo; Gallinella Giorgio

来源：Analytical and Bioanalytical Chemistry, 2013, 405(2-3): 941-949.

DOI：10.1007/s00216-012-6372-1

• 5-Bromo-2 %26apos
• -deoxyuridine
• Chemiluminescence
• Parvovirus B19
• Chemiluminescence microscope imaging assay
• Chemiluminescence dot-blot assay

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%26apos;-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 mu M) 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 x 10(6) B19 genome copies, and the linear range extending up to 5 x 10(8) copies make the method suitable to evaluate the amounts of B19 DNA produced throughout a replicative viral cycle.

• 出版日期2013-1