Fast and reliable detection of viable E. coli bacteria combining direct viability PCR and a novel microarray-based dtection approach
in: Applied and Environmental Microbiology (2019)
We established an innovative approach including a direct, viability and nested PCR for the 30 rapid and reliable identification of the fecal indicator organism Escherichia coli (E. coli) in 31 water samples. Direct PCR enabled the successful amplification of the target uidA gene 32 omitting a prior DNA isolation or purification step. Furthermore, we applied viability PCR (v-33 PCR) to ensure the detection of the only relevant viable bacteria cells. The principle based on 34 propidium monoazide (PMA), a selective nucleic acid intercalating dye, which binds to 35 accessible DNA of dead bacteria cells and consequently allows the discrimination between 36 viable and dead E. coli cells. To gain a high specificity direct v-PCR was followed by a nested 37 PCR step. The resulting amplicons were analysed by a rapid 30 min microarray-based DNA 38 hybridization assay for the species-specific DNA detection of E. coli. A positive signal 39 appeared by enzymatically generated silver nanoparticle deposits, which served as robust 40 endpoint signals allowing an immediate visual readout. The presented novel protocol 41 allowed the detection of 1×101 viable E. coli cells.