| Autor: |
Lydia Lehniger, Anne Rudloff, Sibyll Pollok, Norman Große, Kristin Wessel, Monique Brendel, Jürgen Popp, Karina Weber |
| Jazyk: |
angličtina |
| Rok vydání: |
2021 |
| Předmět: |
|
| Zdroj: |
Chemosensors, Vol 9, Iss 12, p 357 (2021) |
| Druh dokumentu: |
article |
| ISSN: |
2227-9040 |
| DOI: |
10.3390/chemosensors9120357 |
| Popis: |
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. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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