title: Development of an Escherichia coli K12-specific quantitative polymerase chain reaction assay and DNA isolation suited to biofilms associated with iron drinking water pipe corrosion products.
abstract: A quantitative polymerase chain reaction assay (115 bp amplicon) specific to Escherichia coli K12 with an ABI(TM) internal control was developed based on sequence data encoding the rfb gene cluster. Assay specificity was evaluated using three E. coli K12 strains (ATCC W3110, MG1655 & DH1), 24 non-K12 E. coli and 23 bacterial genera. The biofilm detection limit was 10(3) colony-forming units (CFU) E. coli K12 mL(-1), but required a modified protocol, which included a bio-blocker Pseudomonas aeruginosa with ethylenediaminetetraacetic acid buffered to pH 5 prior to cell lysis/DNA extraction. The novel protocol yielded the same sensitivity for drinking water biofilms associated with Fe3O4 (magnetite)-coated SiO2 (quartz) grains and biofilm-surface iron corrosion products from a drinking water distribution system. The novel DNA extraction protocol and specific E. coli K12 assay are sensitive and robust enough for detection and quantification within iron drinking water pipe biofilms, and are particularly well suited for studying enteric bacterial interactions within biofilms.
authors.affiliation: US Environmental Protection Agency ORD, NERL, MCEARD, Cincinnati, OH 45268, USA E-mail: Lu.Jingrang@epa.gov; jingrang_lu@yahoo.com.
authors.forename: Jingrang
authors.initials: J
authors.lastname: Lu
authors.affiliation: ORISE, US Environmental Protection Agency ORD, NERL, MCEARD, Cincinnati, OH 45268, USA and Department of Geology, University of Cincinnati, OH, 45221-0013, USA.
authors.forename: Tammie L
authors.initials: TL
authors.lastname: Gerke
authors.affiliation: US Environmental Protection Agency ORD, NERL, MCEARD, Cincinnati, OH 45268, USA E-mail: Lu.Jingrang@epa.gov; jingrang_lu@yahoo.com; Dynamac Inc., Cincinnati, OH 45268, USA.
authors.forename: Helen Y
authors.initials: HY
authors.lastname: Buse
authors.affiliation: US Environmental Protection Agency ORD, NERL, MCEARD, Cincinnati, OH 45268, USA E-mail: Lu.Jingrang@epa.gov; jingrang_lu@yahoo.com; School of Public Health, University of Alberta, AB T6G 2G7, Canada.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Ashbolt
mesh_terms: D018441:Biofilms / Q000254:growth & development*; D003343:Corrosion; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D060766:Drinking Water / Q000382:microbiology*; D048168:Escherichia coli K12 / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D007501:Iron / Q000378:metabolism*; D057230:Limit of Detection; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D014881:Water Supply / Q000032:analysis*
chemical_list: D004269:DNA, Bacterial; D060766:Drinking Water; D007501:Iron