REMOVAL OF PCR INHIBITORS FROM HUMAN FECAL SAMPLES THROUGH THE USE OFAN AQUEOUS 2-PHASE SYSTEM FOR SAMPLE PREPARATION PRIOR TO PCR

A sample preparation method based on a two-phase aqueous polymer syste m, composed of 8% (w/w) polyethylene glycol 4000 and 11% (w/w) dextran 40, was employed to remove PCR-inhibitory substances from human faece s prior to PCR. The majority of the PCR-inhibitory substances, includi ng bile salts, were shown to be distributed in the polyethylene glycol -rich top phase, whereas target bacteria were detected by PCR in the d extran-rich bottom phase. The efficiency of the aqueous two-phase syst em in removing PCR-inhibitory substances from faeces was evaluated wit h a standardised PCR assay containing different concentrations of pure Helicobacter pylori DNA. The detection level for a pure water solutio n 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 time s 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-tre ated homogenate in the aqueous two-phase system, the detection level w as 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 aque ous two-phase system was applied to faecal samples inoculated with H. pylori cells. The sensitivity of the PCR assay after extraction was fo und to be 40 times above the detection level of H. pylori in a pure wa ter solution. The target bacteria were detected directly by PCR in the dextran-rich bottom phase. Furthermore, most of the H. pylori cells w ere shown to be present in the dextran-rich bottom phase. (C) 1997 Els evier Science B.V.