title: Evaluation of inhibition and cross-reaction effects on real-time PCR applied to the total DNA of wastewater samples for the quantification of bacterial antibiotic resistance genes and taxon-specific targets.
abstract: In order to evaluate the applicability of six primer and probe sets for TaqMan real-time RCR on DNA of wastewater samples, effects of the sample matrix and DNA background on target quantification were studied with respect to differences between functional genes and taxonomically used rDNA targets. Primer/probe assays for real-time PCR (TaqMan) designed to quantitatively detect the antibiotic resistance genes bla(VIM), vanA, ampC, mecA, and taxon-specific 23S rDNA sequences for Pseudomonas aeruginosa and Enterococcus faecium/faecalis were tested for their sensitivity and amplification robustness. The amplification of their gene targets in DNA extracts of wastewater ("wastewater DNA") and reference strains ("reference DNA") was compared with their amplification in "model DNA" which was composed of wastewater DNA and reference DNA. Target detection was quantifiable along up to seven decimal orders of magnitude. For the detection of the resistance genes bla(VIM), vanA, ampC, and mecA as well as the enterococci directed PCR only weak or no inhibition due to the impurities or wastewater DNA matrix were demonstrated for the applied target concentrations. The taxonomically applied detection system for the quantification of P. aeruginosa showed a limited performance. For the analysis of the amplification dynamics of possibly similar nucleotide sequences of organisms related to P. aeruginosa a SYBR Green assay was employed. Competitive amplification of similar sequences was identified to be a major mechanism of reduced sensitivity. Hence, the primers were modified for an optimised detection. With the resulting reduction of cross reactions an increased sensitivity was achieved for the detection and quantification of P. aeruginosa in wastewater DNA.
authors.affiliation: Forschungszentrum Karlsruhe GmbH, Institute for Technical Chemistry, Water Technology and Geotechnology Division, Department of Environmental Microbiology, PO Box 3640, D-76021 Karlsruhe, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Volkmann
authors.forename: Thomas
authors.initials: T
authors.lastname: Schwartz
authors.forename: Silke
authors.initials: S
authors.lastname: Kirchen
authors.forename: Carmen
authors.initials: C
authors.lastname: Stofer
authors.forename: Ursula
authors.initials: U
authors.lastname: Obst
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification*; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D004351:Drug Resistance / Q000235:genetics*; D004754:Enterobacter / Q000235:genetics / Q000302:isolation & purification; D004777:Environment; D016106:Methicillin Resistance; D016133:Polymerase Chain Reaction / Q000379:methods; D013211:Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification; D014865:Waste Disposal, Fluid; D014867:Water / Q000032:analysis*; D014871:Water Microbiology
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D014867:Water