ABSTRACT
Microbial water quality assessment currently relies on cultivation-based methods. Nucleic acid-based techniques such as quantitative PCR (qPCR) enable more rapid and specific detection of target organisms and propidium monoazide (PMA) treatment facilitates the exclusion of false positive results caused by DNA from dead cells. Established molecular assays (qPCR and PMA-qPCR) for legally defined microbial quality parameters (Escherichia coli, Enterococcus spp. and Pseudomonas aeruginosa) and indicator organism group of coliforms (implemented on the molecular detection of Enterobacteriaceae) were comparatively evaluated to conventional microbiological methods. The evaluation of an extended set of drinking and process water samples showed that PMA-qPCR for E. coli, Enterococcus spp. and P. aeruginosa resulted in higher specificity because substantial or complete reduction of false positive signals in comparison to qPCR were obtained. Complete compliance to reference method was achieved for E. coli PMA-qPCR and 100% specificity for Enterococcus spp. and P. aeruginosa in the evaluation of process water samples. A major challenge remained in sensitivity of the assays, exhibited through false negative results (7-23%), which is presumably due to insufficient sample preparation (i.e. concentration of bacteria and DNA extraction), rather than the qPCR limit of detection. For the detection of the indicator group of coliforms, the evaluation study revealed that the utilization of alternative molecular assays based on the taxonomic group of Enterobacteriaceae was not adequate. Given the careful optimization of the sensitivity, the highly specific PMA-qPCR could be a valuable tool for rapid detection of hygienic parameters such as E. coli, Enterococcus spp. and P. aeruginosa.
Subject(s)
Azides/pharmacology , Drinking Water/microbiology , Polymerase Chain Reaction/methods , Propidium/analogs & derivatives , Water Microbiology , Water Quality/standards , Austria , DNA Primers/genetics , Propidium/pharmacologyABSTRACT
Diversity Arrays Technology (DArT) was applied to differentiate between S. enterica serovar Enteritidis and Typhimurium strains, respectively. Ten and eleven, mainly phage and plasmid-related markers were identified for serovars Enteritidis and Typhimurium. In combination, these markers can be used for subtyping among and within phage types.
Subject(s)
Bacteriophage Typing/methods , Bacteriophages/chemistry , Salmonella enteritidis/isolation & purification , Salmonella typhimurium/isolation & purification , Viral Proteins/analysis , Humans , Molecular Sequence Data , Phylogeny , Salmonella Infections/microbiology , Salmonella enteritidis/chemistry , Salmonella enteritidis/classification , Salmonella enteritidis/virology , Salmonella typhimurium/chemistry , Salmonella typhimurium/classification , Salmonella typhimurium/virologyABSTRACT
A method was developed for the mRNA-based application of microbial diagnostic microarrays to detect active microbial populations. DNA- and mRNA-based analyses of environmental samples were compared and confirmed via quantitative PCR. Results indicated that mRNA-based microarray analyses may provide additional information on the composition and functioning of microbial communities.
