Removal of PCR inhibitors from human faecal samples through the use of an aqueous two-phase system for sample preparation prior to PCR

A sample preparation method based on a two-phase aqueous polymer system, composed of 8% (w/w) polyethylene glycol 4000 and 11% (w/w) dextran 40, was employed to remove PCR-inhibitory substances from human faeces prior to PCR. The majority of the PCR-inhibitory substances, including bile salts, were shown to be distributed in the polyethylene glycol-rich top phase, whereas target bacteria were detected by PCR in the dextran-rich bottom phase. The efficiency of the aqueous two-phase system in removing PCR-inhibitory substances from faeces was evaluated with a standardised PCR assay containing different concentrations of pure Helicobacter pylori DNA. The detection level for a pure water solution of DNA was in the range 10(-12) to 10(-13) g DNA per reaction tube. Untreated faecal homogenate totally inhibited PCR even after 1000 times dilution in water. A positive PCR result was obtained when 10(-6) g H. pylori DNA was present in a 50 mu l reaction mixture containing 10 CLI homogenised faeces, which had been briefly centrifuged, boiled and diluted 100 times in water. After extraction of an undiluted heat-treated homogenate in the aqueous two-phase system, the detection level was lowered to the range 10(-10) to 10(-11) g DNA. When the aqueous two-phase system was evaluated on five faecal samples the detection level was decreased by three to five orders of magnitude. Finally, the aqueous two-phase system was applied to faecal samples inoculated with H. pylori cells. The sensitivity of the PCR assay after extraction was found to be 40 times above the detection level of H. pylori in a pure water solution. The target bacteria were detected directly by PCR in the dextran-rich bottom phase. Furthermore, most of the H. pylori cells were shown to be present in the dextran-rich bottom phase. (C) 1997 Elsevier Science B.V.