Scottish Government

METHODS

Quality issues

The presence of H. pyloriDNA in drinking water samples has many important consequences. Therefore it was important that all appropriate measures were undertaken to ensure the reliability of results. In the course of our experiments we have:

• Performed pre- and post- PCR processing in separate laboratories with separate consumables and reagents to avoid cross-contamination
• Processed DNA extracted from H. pylori strains alongside the sample materials to provide a positive control
• Processed DNA from non-pylori Helicobacter spp. to verify the specificity of the assays for H. pyloriDNA
• Processed sample blanks (consisting of ultra-pure water only) to serve as an indicator of contamination
• Accepted sample materials that yield PCR amplicons to be subsequently sequenced and identified as H. pyloriDNA as genuine only if each of the control assays performs as expected

Biofilm samples

Biofilm samples were supplied by Scottish Water (Edinburgh, UK). They consisted of biofilm material removed from pipe sections extracted from the drinking water distribution network in Scotland. The exact geographical origins of these samples and their storage history were not disclosed. They were supplied with identification codes and information on the presence/absence of H. pyloriDNA that had been obtained in previous studies (Table 2). Samples from five sites were originally identified as possibly containing H. pyloriDNA. Samples from three additional sites that were originally negative for H. pyloriDNA were included as a control. On arrival at RHSC (Glasgow, UK) one of the biofilm samples was missing (sample 719). The sample was requested from Scottish Water but did not arrive. A summary of the biofilm samples is provided in Table 2.

Table 2: Sample identity and corresponding Helicobacter pyloriPCR data for the biofilm samples received from Scottish Water (numbers correspond to the sample site [not disclosed], letters correspond to multiple samples from each site)

Extraction of DNA from biofilm samples

DNA was extracted using the Ultra Clean Soil DNA Kit according to the manufacturers instructions (Cambio Ltd.,Cambridge, UK). Briefly, water samples were added to a bead beating tube and vortexed to lyse the cells. DNA from these lysed cells was then bound to a filter membrane and the DNA eluted into TE buffer (10 mM Tris, 1 mM EDTA). DNA samples were stored at -20ºC until analysis.

Validation of the Extraction methodology

Serial dilutions of H. pyloriDNA from neat to 1x10 ^-5 were prepared in tap water (collected from the laboratory at the Division of Developmental Medicine at Yorkhill Hospitals, Glasgow UK) and subjected to DNA extraction as described above. The H. pylori isolate used was harvested (by gastroscopy) from the stomach of an H. pylori infected volunteer who presented at Gartnavel General Hospital (Glasgow) with Duodenal ulceration. The identity of the isolate was determined by standard classical and molecular methods as described in Ndip et al (2003). Serial dilutions of the H. pyloriDNA were also prepared in sterile ultrapure water. Comparison of the DNA recovery from both sets of DNA samples was used to determine whether the DNA purification protocol resulted in a loss of target DNA.

DNA samples were then subjected to PCR analysis. Each PCR reaction contained 1 x reaction buffer, 2.5mM MgCl ₂, 200µM d NTPs, forward and reverse primers at 1µM, 1.25U of a Hotstart DNA polymerase, and 10µl of target DNA (final reaction volume of 50µl). Primers were synthesised by Sigma- genosys Ltd. (Haverhill, UK) and were Act1 (5'- CTTGCTAGAGTGCTGATT A-3') and Act2 (5'- TCCCACACTCTAGAATAG T-3') and amplified 527 base pairs of the 16S r RNA gene of H. pylori (Thoreson et al 1995). The negative control consisted of sterile ultrapure water instead of template DNA.

Thermal cycling was performed with an initial melting at 94° C for 15 min and 30 cycles of denaturation at 94° C for one min, annealing at 60° C for one min and extension at 72° C for one min with a final extension step at 72° C for ten min.

All PCR products were analysed by gel electrophoresis in a 3% (w/v) agarose gel (containing 0.6µg ml ^-1 ethidium bromide) prepared in 0.5x Tris acetate EDTA ( TAE) buffer. Gels were run for one hr at 50V (RunOne Electrophoresis cell (Embitec, San Diego, USA) and DNA bands examined under ultra violet illumination.

Determination of the presence of amplifiable Helicobacter pyloriDNA in biofilm samples

An H. pylori-specific sequencing PCR directed against the 16S r RNA gene was used to determine whether the biofilm samples contained amplifiable H. pyloriDNA. DNA was extracted from biofilm samples as described above and subjected to PCR analysis as follows. Each PCR reaction contained 1 x reaction buffer, 3mM MgCl ₂, 200µM d NTPs, forward and reverse primers at 1µM, 1.25U of a Hotstart DNA polymerase, and 10µl of target DNA (final reaction volume of 50µl). Primers were synthesised by Sigma- genosys Ltd. (Haverhill, UK) and were HpF (5- GCGACCTGCTGGAACATTAC-3) and HpR (5- CGTTAGCTGCATTACTGGAGA-3), and amplified 180 base pairs of the 16S r RNA gene of H. pylori (Gramley et al 1999). The positive control consisted of purified H. pyloriDNA and the negative control of sterile ultrapure water.

The specificity of the H. pyloriPCR assay was determined by testing four non-pylori Helicobacters and one Campylobacter sp. ( C. jejuni). Bacterial isolates were from the gastrointestinal bacterial culture collection of Dr William MacKay (Division of Developmental Medicine, University of Glasgow, UK). They were cultivated on columbia blood agar base (Oxoid, Hants, UK) supplemented with 7% defibrinated horse blood ( TCS Biosciences, Buckingham, UK) for seven days under microaerobic conditions (37ºC) in gas jars (Becton Dickinson, Oxford, UK). The total biomass was harvested and resuspended in PBS buffer (0.01M phosphate buffer, 0.0027M potassium chloride, 0.317M sodium chloride [pH 7.4]) and enumerated by microscopy (at x 1000 magnification [phase contrast]) under oil emersion on a Leica Axioscope Microscope (Leica Microsystems, Milton Keynes, UK) using a cell-counting chamber (Sigma-Aldrich, Dorset, UK) according to the manufacturers instructions.

For the determination of sensitivity, H. pyloriDNA was assayed for concentration by spectrophotometry (260nm) and serially diluted in sterile ultrapure water (Sigma-Aldrich, Dorset, UK). H. PyloriPCR analysis was performed on each dilution. The sensitivity of the PCR assay was defined as the lowest concentration of DNA that resulted in PCR amplification. Results were reported in fmoles where one H. pylori genome was ~1.7fmol (Gramley et al 1999).

Thermal cycling with performed with an initial melting at 94° C for 15 min and 28 cycles of denaturation at 94° C for one min, annealing at 62° C for one min and extension at 72° C for one min with a final extension step at 72° C for ten min.

All PCR products were analysed by gel electrophoresis in a 3% (w/v) agarose gel (containing 0.6µg ml ^-1 ethidium bromide) prepared in 0.5x Tris acetate EDTA ( TAE) buffer. Gels were run for one hr at 50V (RunOne Electrophoresis cell (Embitec, San Diego, USA) and DNA bands examined under ultra violet illumination.

Identification of Helicobacter pyloriDNA by sequence analysis

PCR reactions that yielded amplified DNA were subjected to sequence analysis for definitive identification. Sequencing was performed using an automated system (Mega BACE1000 - 96 capillary system) and Big Dye technologies to produce a colour electropherogram for each sample. These data were then submitted to the BLASTDNA sequence database ( http://www.ncbi.nlm.nih.gov/BLAST/).

The identity of the sequenced DNA was determined by matching it to published sequences in the BLAST database. Matches were reported as a percentage.