USE OF AQUEOUS 2-PHASE SYSTEMS IN SAMPLE PREPARATION FOR POLYMERASE CHAIN REACTION-BASED DETECTION OF MICROORGANISMS

An aqueous two-phase system, consisting of poly(ethylene glycol) (PEG) and dextran, was employed to separate polymerase chain reaction (PCR) -inhibitory substances from bacterial cells. The PCR inhibition of fou r soft cheeses was examined and three of them were found to be strongl y PCR-inhibitory. Extraction of the PCR-inhibitory soft cheeses inocul ated with Listeria monocytogenes in an aqueous two-phase system contai ning 8% (w/w) PEG 4000 and 8% (w/w) dextran 500, was found to lower th e PCR detection level of L. monocytogenes by more than four orders of magnitude in two of the cheeses compared to the case where no such sam ple pretreatment was performed. Depending on the type of cheese used, the PCR-inhibitory factors were found to be enriched in either the top or bottom phase in the aqueous two-phase system. These results show t hat different soft cheeses contain different types and amounts of PCR- inhibitory substances.