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Fast‐Track, One‐Step E. coli Detection: A Miniaturized Hydrogel Array Permits Specific Direct PCR and DNA Hybridization while Amplification
Author(s) -
Beyer Antje,
Pollok Sibyll,
Rudloff Anne,
CiallaMay Dana,
Weber Karina,
Popp Jürgen
Publication year - 2016
Publication title -
macromolecular bioscience
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.924
H-Index - 105
eISSN - 1616-5195
pISSN - 1616-5187
DOI - 10.1002/mabi.201600098
Subject(s) - polymerase chain reaction , pipette , dna , chemistry , fluorescence , fluorescence microscope , detection limit , dna extraction , microbiology and biotechnology , biophysics , materials science , chromatography , biochemistry , gene , optics , biology , physics
A timesaving and convenient method for bacterial detection based on one‐step, one‐tube deoxyribonucleic acid (DNA) hybridization on hydrogel array while target gene amplification is described. The hydrogel array is generated by a fast one‐pot synthesis, where N,N ′‐dimethylacrylamide/polyethyleneglycol(PEG 1900 )‐bisacrylamide mixture polymerizes via radical photoinitiation by visible light within 20 min concomitant with in situ capture probe immobilization. These DNA‐functionalized hydrogel droplets arrayed on a planar glass surface are placed in the polymerase chain reaction (PCR) mixture during the thermal amplification cycles. The bacterial cells can be implemented in a direct PCR reaction, omitting the need for prior template DNA extraction. The resulting fluorescence signal is immediately detectable after the end of the PCR (1 h) following one short washing step by microscopy. Therefore a valid signal can be reached within 1.5 h including 10 min for pipetting and placement of the tubes and chips. The performance of this novel hydrogel DNA array was successfully proven with varying cell numbers down to a limit of 10 1 Escherichia coli cells.