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A sample treatment method based on an aqueous two-phase system containing polyethylene glycol and dextran was developed for enhancing sensitivity in the detection of Listeria monocytogenes in soft cheese with PCR. The results suggest that the improved detection sensitivity following partitioning of the cheese homogenate in an aqueous two-phase system may be due to partitioning of the PCR inhibitors to the polyethylene glycol phase. PCR is one of the most promising techniques for rapid detection of microorganisms in food. However, the usefulness of PCR for detection of microbes in soft cheeses and other complex samples is limited by the presence of factors that inhibit PCR (7, 10, 14). The aim of the present study was to investigate whether an aqueous two-phase system (1, 12) can provide the basis for a sample preparation method which separates PCR-inhibitory factors from the bacteria in a food sample rather than vice versa, which is the common approach (2, 5, 13). The approach taken was to employ PCR for direct detection of Listeria monocytogenes cells after their extraction in the aqueous two-phase system. PCR conditions. The PCR assay comprised two DNA amplification steps (Fig. 1). Primers LM2 (5'-CC'TTIGACCACT CTGGAGACAGAGC-3') and LM1 (5'-GGAGCTAATCCC ATAAAACTA-3') were designed on the basis of published nucleotide sequences of 16S rRNA genes (4). The first step involved amplification with oligonucleotides LM2 and ru8 (5'-AAGGAGGTGATCCA[G/A]CCGCA[G/C][G/C]TTC-3' [8]) for 30 cycles, and the second amplification step was performed with oligonucleotides LM1 and ru8 for 30 cycles, the latter after 1:10 dilution of the product obtained from the first PCR incubation. The 553and 275-bp PCR products were visualized by ethidium bromide-stained 1.5% agarose gel electrophoresis (11). The PCR mixture (50 ,u) contained 1.5 U of Taq DNA polymerase (Boehringer GmbH, Mannheim, Germany), 1X PCR buffer (Boehringer), each primer at 0.5 ,uM, and each of the deoxynucleoside triphosphates at 0.2 mM. The sample volume was 10 RI. The reaction tubes were placed in a thermal

A polymerase chain reaction (PCR) based diagnostic assay has been developed for the simultaneous detection in cerebrospinal fluid (CSF) of Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, Streptococcus agalactiae, Listeria monocytogenes and bacteria in general. In the present communication we describe the design of primers for S. pneumoniae, S. agalactiae, and L. monocytogenes, and a general PCR protocol for the assay. The diagnostic outcome is presented for a small collection of CSF specimens including 2 samples from patients with culture-negative purulent meningitis.