title: Highly rapid amplification-free and quantitative DNA imaging assay.
abstract: There is an urgent need for rapid and highly sensitive detection of pathogen-derived DNA in a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a 'sample-in-result-out' mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5 kbp target DNA fragments of Micrococcus luteus in small sample volumes of 20 μL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of ~5 pM and a linear detection range spanning three orders of magnitude.
authors.affiliation: Department of Biotechnology & Biophysics, Biocenter, Julius-Maximilians-University Würzburg, Am Hubland, 97074 Würzburg, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Klamp
authors.forename: Marta
authors.initials: M
authors.lastname: Camps
authors.forename: Benjamin
authors.initials: B
authors.lastname: Nieto
authors.forename: Francesc
authors.initials: F
authors.lastname: Guasch
authors.forename: Rohan T
authors.initials: RT
authors.lastname: Ranasinghe
authors.forename: Jens
authors.initials: J
authors.lastname: Wiedemann
authors.forename: Zdeněk
authors.initials: Z
authors.lastname: Petrášek
authors.forename: Petra
authors.initials: P
authors.lastname: Schwille
authors.forename: David
authors.initials: D
authors.lastname: Klenerman
authors.forename: Markus
authors.initials: M
authors.lastname: Sauer
mesh_terms: D004247:DNA / Q000032:analysis*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D004247:DNA