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.

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.

Journal of water and health (2014-12-05)

Jingrang Lu, Tammie L Gerke, Helen Y Buse, Nicholas J Ashbolt

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.

MATERIALS

Product Number

Brand

Product Description

IRMM524B

Iron, IRMM^®, certified reference material, 0.5 mm wire

267945

Sigma-Aldrich

Iron, chips, 99.98% trace metals basis

44890

Sigma-Aldrich

Iron, puriss. p.a., carbonyl-Iron powder, low in magnesium and manganese compounds, ≥99.5% (RT)

209309

Sigma-Aldrich

Iron, powder, −325 mesh, 97%

356808

Sigma-Aldrich

Iron, foil, thickness 0.1 mm, ≥99.9% trace metals basis

GF63891726

Iron, insulated wire, 10m, conductor diameter 0.25mm, insulation thickness 0.01mm, polyester insulation

GF33572606

Iron, tube, 500mm, outside diameter 5.0mm, inside diameter 4.5mm, wall thickness 0.25mm, as drawn, 99.5%

GF50403440

Iron, tube, 1000mm, outside diameter 12.0mm, inside diameter 10mm, wall thickness 1.0mm, annealed, 99.5%

GF83953952

Iron, tube, 200mm, outside diameter 2.38mm, inside diameter 1.6mm, wall thickness 0.39mm, as drawn, 99.8+%

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Iron, tube, 1000mm, outside diameter 12.0mm, inside diameter 9mm, wall thickness 1.5mm, annealed, 99.5%

GF21950197

Iron, tube, 1000mm, outside diameter 3.18mm, inside diameter 1.96mm, wall thickness 0.61mm, as drawn, 99.5%

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Iron, tube, 1000mm, outside diameter 25.0mm, inside diameter 21mm, wall thickness 2mm, annealed, 99.5%

GF82933511

Iron, tube, 1000mm, outside diameter 3.2mm, inside diameter 2.2mm, wall thickness 0.5mm, as drawn, 99.8%

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GF93209328

Iron, tube, 200mm, outside diameter 8.0mm, inside diameter 5mm, wall thickness 1.5mm, annealed, 99.5%

GF39321390

Iron, tube, 100mm, outside diameter 10.6mm, inside diameter 10.0mm, wall thickness 0.3mm, as drawn, 99.8+%

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GF31151887

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Iron, tube, 1000mm, outside diameter 5.0mm, inside diameter 4.5mm, wall thickness 0.25mm, as drawn, 99.5%

GF17042082

Iron, tube, 200mm, outside diameter 4.0mm, inside diameter 3.6mm, wall thickness 0.2mm, as drawn, 99.5%

GF61545674

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GF30656077

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GF04812599

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GF38919279

Iron, tube, 200mm, outside diameter 20.0mm, inside diameter 16mm, wall thickness 2.0mm, annealed, 99.5%

GF40975601

Iron, tube, 500mm, outside diameter 3.18mm, inside diameter 1.96mm, wall thickness 0.61mm, as drawn, 99.5%