title: Homogenous M13 bacteriophage quantification assay using switchable lanthanide fluorescence probes.
abstract: We have developed a rapid and reliable bacteriophage quantification method based on measurement of phage single-stranded DNA (ssDNA) using switchable lanthanide chelate complementation probes. One oligonucleotide probe contains a non-fluorescent lanthanide ion carrier chelate and another probe is labeled with a light absorbing antenna ligand. Hybridization of the non-fluorescent complementation probes in adjacent positions on the released bacteriophage ssDNA leads to high local concentrations of the lanthanide ion carrier chelate and the antenna ligand, inducing formation of a fluorescent lanthanide chelate complex. This method enables monitoring of bacteriophage titers in a 20 min assay with a dynamic range of 10(9)-10(12) cfu/mL in a microtiter well format. While designed for titering filamentous bacteriophage used in phage display, our method also could be implemented in virological research as a tool to analyze ssDNA virus reproduction.
authors.affiliation: Department of Biochemistry and Food Chemistry, Division of Biotechnology, University of Turku, Finland. artule@utu.fi
authors.forename: Ari
authors.initials: A
authors.lastname: Lehmusvuori
authors.forename: Julius
authors.initials: J
authors.lastname: Manninen
authors.forename: Tuomas
authors.initials: T
authors.lastname: Huovinen
authors.forename: Tero
authors.initials: T
authors.lastname: Soukka
authors.forename: Urpo
authors.initials: U
authors.lastname: Lamminmäki
mesh_terms: D017104:Bacteriophage M13 / Q000235:genetics*; D001709:Biotechnology; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D057230:Limit of Detection; D008957:Models, Genetic; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics
chemical_list: D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D028581:Lanthanoid Series Elements; D015345:Oligonucleotide Probes; D057127:Single-Chain Antibodies