A Model System for Sensitive Detection of Viable <i>E. coli</i> Bacteria Combining Direct Viability PCR and a Novel Microarray-Based Detection Approach
Lydia Lehniger,
Anne Rudloff,
Sibyll Pollok,
Norman Große,
Kristin Wessel,
Monique Brendel,
Jürgen Popp,
Karina Weber
Affiliations
Lydia Lehniger
Leibniz Institute of Photonic Technology, Member of the Research Alliance “Leibniz Health Technologies”, Albert-Einstein-Straße 9, 07745 Jena, Germany
Anne Rudloff
Leibniz Institute of Photonic Technology, Member of the Research Alliance “Leibniz Health Technologies”, Albert-Einstein-Straße 9, 07745 Jena, Germany
Sibyll Pollok
Leibniz Institute of Photonic Technology, Member of the Research Alliance “Leibniz Health Technologies”, Albert-Einstein-Straße 9, 07745 Jena, Germany
Norman Große
Leibniz Institute of Photonic Technology, Member of the Research Alliance “Leibniz Health Technologies”, Albert-Einstein-Straße 9, 07745 Jena, Germany
Kristin Wessel
IST Innuscreen GmbH, Robert-Rössle-Straße 10, 13125 Berlin, Germany
Monique Brendel
IST Innuscreen GmbH, Robert-Rössle-Straße 10, 13125 Berlin, Germany
Jürgen Popp
Leibniz Institute of Photonic Technology, Member of the Research Alliance “Leibniz Health Technologies”, Albert-Einstein-Straße 9, 07745 Jena, Germany
Karina Weber
Leibniz Institute of Photonic Technology, Member of the Research Alliance “Leibniz Health Technologies”, Albert-Einstein-Straße 9, 07745 Jena, Germany
We established an innovative approach that included direct, viability, and nested PCR for rapid and reliable identification of the fecal indicator organism Escherichia coli (E. coli). Direct PCR enabled successful amplification of the target uidA gene, omitting a prior DNA isolation or purification step. Furthermore, we applied viability PCR (v-PCR) to ensure the detection of only relevant viable bacterial cells. The principle involves the binding of propidium monoazide (PMA), a selective nucleic acid intercalating dye, to accessible DNA of heat killed bacteria cells and, consequently, allows viable and heat killed E. coli cells to be discriminated. To ensure high sensitivity, direct v-PCR was followed by a nested PCR step. The resulting amplicons were analyzed by a rapid 30 min microarray-based DNA hybridization assay for species-specific DNA detection of E. coli. A positive signal was indicated by enzymatically generated silver nanoparticle deposits, which served as robust endpoint signals allowing an immediate visual readout. The presented novel protocol allows the detection of 1 × 101 viable E. coli cells per PCR run.
